Biosynthesis pathway for prenol in a recombinant microorganism

ABSTRACT

The present invention concerns a method for the biological preparation of prenol comprising culturing a microorganism genetically modified for the bioproduction of prenol, wherein the microorganism comprises a metabolic pathway for conversion of 3-methylcrotonyl-CoA into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme.

The present invention concerns a method for the biological preparation of prenol comprising culturing a microorganism genetically modified for the bioproduction of prenol, wherein the microorganism comprises a metabolic pathway for conversion of 3-methylcrotonyl-CoA into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme.

INTRODUCTION

Prenol or 3-methyl-2-buten-1-ol (number CAS 556-82-1), has the following formula:

Prenol is a natural alcohol that occurs naturally in citrus fruits, cranberry, bilberry, currants, grapes, raspberry, blackberry, tomato, white bread, hop oil, coffee, arctic bramble, cloudberry and passion fruit.

Prenol is under the form of a clear, colorless oil that is reasonably soluble in water and miscible with most common organic solvents. It has a fruity odor and is used as an aroma compound, in pharmaceuticals and in perfumery. It is usually manufactured industrially, in particular by BASF.

Advantageously, prenol can be converted into isoprene, a compound of high importance for industry since it is the basis for the synthesis of synthetic rubber.

Isoprene (short for isoterpene) or 2-methyl-1,3-butadiene (Numero CAS 78-79-5) is an organic compound having the formula CH2=(CH3)CH═CH₂. Under standard conditions it is a colorless liquid. However, this compound is highly volatile because of its low boiling point. Isoprene is the monomer of natural rubber and also a common structure motif to an immense variety of other naturally occurring compounds, collectively termed the isoprenoids.

About 95% of isoprene production is used to produce cis-1,4-polyisoprene—a synthetic version of natural rubber. Isoprene is also copolymerized for use as a synthetic elastomer in other products such as footwear, mechanical products, medical products, sporting goods, and latex.

Isoprene was first isolated by thermal decomposition of natural rubber. It is most readily available industrially as a by-product of the thermal cracking of naphtha or oil, as a side product in the production of ethylene. While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming.

Isoprene is naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate pathway and the non-mevalonate pathway. However, the yield of isoprene from naturally-occurring organisms is commercially unattractive.

Thus, more economical methods for producing isoprene are needed. In particular, methods that produce isoprene at rates, titers, and purity that are sufficient to meet the demands of a robust commercial process are desirable. Also desired are systems for producing isoprene from inexpensive starting materials.

PRIOR ART

Although the main industrial way for producing prenol is chemical synthesis, some biosynthetic pathways have been identified recently.

The patent application WO 2009/006429 provides a method for producing prenol with a genetically modified cell expressing a first enzyme capable of catalysing the dephosphorylation of the intermediate products: isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP).

Concerning the isoprene synthesis, methods for fermentative production have been described. In particular, WO 2009/076676 describes cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide. Two biosynthesis pathways are proposed.

The patent application WO 2010/031076 describes the conversion of prenyl derivatives, such as prenol, into isoprene. Said method comprises: a) culturing cells for producing prenol, wherein the cells comprise a heterologous isoprene synthase polypeptide, b) recovering prenol, and c) dehydrating or decarboxylating prenol to produce isoprene. The cells may further comprise: an IDI polypeptide, an MVA pathway enzyme, and a DXP pathway enzyme.

DESCRIPTION OF THE INVENTION

The present invention is related to a new biosynthesis pathway for prenol in a recombinant microorganism. This biosynthesis pathway is characterized by the intermediate product, that is 3-methylcrotonyl-CoA (number CAS 6247-62-7), and that is converted into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme.

In particular, the enzyme capable of converting 3-methylcrotonyl-CoA into prenol is the alcohol-aldehyde dehydrogenase enzyme AdhE, that is heterologous to the recombinant microorganism, and that is preferentially issued from Clostridium acetobutylicum.

According to the invention, the intermediate product 3-methylcrotonyl-CoA can be obtained from two different metabolic pathways:

-   -   1) From the degradation pathway of leucine, starting from a         condensation of pyruvate and acetyl-CoA into         4-methyl-2-oxopentanoate, converted into 3-methylbutanoyl-CoA         and then into 3-methylcrotonyl-CoA. Here, this pathway is named         leucine pathway,     -   2) From the mevalonate biosynthesis pathway, the precursor         3-hydroxy-3-methylglutaryl-CoA being converted into         3-methylglutaconyl-CoA and then into 3-methylcrotonyl-CoA.         Formation of 3-methylcrotonyl-CoA from two acetyl-CoA molecules         has been suggested to be possible in the article from Gogerty         and Bobik (Gogerty and Bobik, 2010). Here, this pathway is named         HMG-CoA pathway.

According to a specific aspect of the invention, the obtained prenol is converted into isoprene by chemical dehydration.

DRAWINGS

FIG. 1. Metabolic pathway for biosynthesis of prenol, from the leucine pathway.

FIG. 2. Metabolic pathway for biosynthesis of prenol, from the HMG-CoA pathway.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting, which will be limited only by the appended claims.

All publications, patents and patent applications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors that are reported in the publications and that might be used in connection with the invention.

Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Prescott et al., (1999) and Sambrook et al., (1989) (2001).

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a microorganism” includes a plurality of such microorganisms, and a reference to “an endogenous gene” is a reference to one or more endogenous genes, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

In the claims that follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

DEFINITIONS

The term “prenol” as used herein refers to 3-methyl-2-buten-1-ol or to a 3,3-dimethylallyl alcohol or DMAPP-ol while the term “isoprenol” refers especially to 3-methyl-3-buten-1-ol or IPP-ol.

3-Methylcrotonyl-CoA or β-Methylcrotonyl-CoA (CAS number 6247-62-7) is an intermediate in the metabolism of leucine. It is usually formed from 3-methylbutanoyl-CoA (also called isovaleryl-coenzyme A) by isovaleryl-coenzyme A dehydrogenase.

The term “aldehyde dehydrogenase” in this invention designates the aldehyde dehydrogenase (CoA-acylating) enzyme which catalyzes the reaction of conversion of an acyl-CoA into an aldehyde.

The term “alcohol dehydrogenase” in this invention designates the enzyme which catalyzes the reaction of conversion of an aldehyde into an alcohol.

Here, the term “AdhE enzyme” refers to a bifunctional enzyme having the two activities aldehyde dehydrogenase and alcohol dehydrogenase.

The terms “activity” and “function” refer to a specific catalytic activity or function of an enzyme, i.e. the biochemical reaction(s) that is (are) catalyzed by this enzyme.

The term “microorganism”, as used herein, refers to a bacterium, yeast or fungus which is not modified artificially.

The term “recombinant microorganism” or “genetically modified microorganism”, as used herein, refers to a microorganism genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction, by deletion or by modification of genetic elements. It can also be transformed by forcing the development and evolution of new metabolic pathways in combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).

A microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. A microorganism may be modified to modulate the expression level of an endogenous gene. The modification or “transformation” of microorganisms with exogenous DNA is a routine task for those skilled in the art.

The term “endogenous gene” means that the gene was present in the microorganism before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, down regulate and/or lower the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.

The term “exogenous gene” means that the gene was introduced into a microorganism, by means well known by the man skilled in the art whereas this gene is not naturally occurring in the microorganism. Exogenous genes can be heterologous or not. A microorganism can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally by plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. These genes may be heterologous or homologous. The term “heterologous gene” means that the gene is derived from a species of microorganism different from the recipient microorganism that expresses it. It refers to a gene which is not naturally occurring in the microorganism.

In the present application, all genes are referenced with their common names and with references that give access to their nucleotidic sequences in the National Center for Biotechnology Information (NCBI) GenBank.

The man skilled in the art knows different means to modulate, and in particular up-regulate, the expression of endogenous genes. For example, a way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.

Another way is to replace the endogenous promoter of a gene with a stronger promoter. These promoters may be homologous or heterologous. It is well within the ability of the person skilled in the art to select appropriate promoters, for example, the promoters Ptrc, Ptac, Plac or the lambda promoter cI are widely used.

Finally, the sequence of exogenous gene may be adapted for its expression in the host microorganism. Indeed, the man skilled in the art knows the notion of codon usage bias and how adapt nucleic sequence for a particular codon usage bias without modify the deduced protein.

The term ‘overexpression’ means in this context that the expression of a gene or an enzyme is increased compared to a non modified microorganism. Increase of expression of an enzyme is obtained by the increase of the expression of a gene encoding said enzyme.

The ‘activity’ of an enzyme is used interchangeably with the term ‘function’ and designates, in the context of the invention, the reaction that is catalyzed by the enzyme.

The terms “encoding” or “coding” refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.

The gene(s) encoding the enzyme(s) can be exogenous or endogenous.

“Attenuation” of genes may be achieved by means and methods known to the man skilled in the art and contains gene deletion by homologous recombination, gene attenuation by insertion of an external element into the gene or gene expression under a weak promoter. The man skilled in the art knows a variety of promoters which exhibit different strength and which promoter to use for a weak genetic expression.

The “fermentation” is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being used, containing at least one simple carbon source, and if necessary co-substrates.

An “appropriate culture medium” designates a medium (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids and vitamins.

As an example of known culture media for E. coli, the culture medium can be of identical or similar composition to an M9 medium (Anderson, 1946), an M63 medium (Miller, 1992) or a medium such as defined by Schaefer et al., (1999).

As another example of culture medium for C. glutamicum, the culture medium can be of identical or similar composition to BMCG medium (Liebl et al., 1989) or to a medium such as described by Riedel et al., (2001).

Those skilled in the art are able to define the culture conditions for the microorganisms according to the invention. In particular the bacteria are fermented at a temperature between 20° C. and 55° C., preferentially between 25° C. and 40° C., and more specifically about 30° C. for C. glutamicum and about 37° C. for E. coli.

The term “carbon source” or “carbon substrate” or “source of carbon” according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a micro-organism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, hemicelluloses and combinations thereof.

Prenol Biosynthesis

The present invention is related to a method for the fermentative production of prenol, comprising culturing a recombinant microorganism in a culture medium comprising a source of carbon, wherein in said microorganism, the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA as intermediate product, that is converted into prenol by the action of an alcohol dehydrogenase enzyme and of an aldehyde dehydrogenase enzyme.

An alcohol dehydrogenase enzyme designates an enzyme catalyzing the following reactions, in one way or in the other: an aldehyde(or a ketone)+NAD(P)H+H+

an alcohol+NAD(P)+

This enzyme always functions with an ‘acceptor’ of hydrogen, such as NAD+, NADP+, or other specific acceptors. The activity of this enzyme is the conversion of an alcohol into an aldehyde, and/or the conversion of an aldehyde into an alcohol with a donor of hydrogen.

Other known names are: aldehyde reductase; ADH; NAD-dependent alcohol dehydrogenase; NADH-alcohol dehydrogenase; primary alcohol dehydrogenase; aldehyde reductase (NADPH); NADP-alcohol dehydrogenase; NADP-aldehyde reductase; NADP-dependent aldehyde reductase; NADPH-aldehyde reductase; NADPH-dependent aldehyde reductase; alcohol dehydrogenase (NADP); the common abbreviation is ADH.

In a specific aspect of the invention, the enzyme having alcohol dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 1:

Gene name Enzyme name Organism adhA alcohol-dehydrogenase adhA from patent WO8704464-A- A. niger Aspergillus niger (EC: 1.1.1.1) ADH1 ADH1 (ALCOHOL DEHYDROGENASE 1); alcohol A. thaliana dehydrogenase adhA alcohol dehydrogenase (EC: 1.1.1.1) B. cereus gbsB choline dehydrogenase (EC: 1.1.1.—) B. subtilis adhB putative oxidoreductase (EC: 1.1.1.—) B. subtilis adhE1 bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. acetobutylicum adhE2 bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. acetobutylicum cgl2537 putative Zn-NADPH:quinone dehydrogenase (EC: 1.1.1.1) C. glutamicum cgl0222 Zn-dependent alcohol dehydrogenase (EC: 1.1.1.1) C. glutamicum cgl2807 Zn-dependent alcohol dehydrogenase (EC: 1.1.1.1) C. glutamicum aad bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. kluyveri (EC: 1.1.1.—1.2.1.—) adhP ethanol-active dehydrogenase/acetaldehyde-active reductase E. coli (EC: 1.1.1.1) yiaY predicted Fe-containing alcohol dehydrogenase, Pfam00465 E. coli family adhE fused acetaldehyde-CoA dehydrogenase/iron-dependent E. coli alcohol dehydrogenase/pyruvate-formate lyase deactivase (EC: 1.2.1.10 1.1.1.1) frmA alcohol dehydrogenase class III/glutathione-dependent E. coli formaldehyde dehydrogenase (EC: 1.1.1.1 1.1.1.284) adhA alcohol dehydrogenase E. faecalis adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase E. faecalis adhC alcohol dehydrogenase class III H. influenzae adhP alcohol dehydrogenase K. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase K. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase L. lactis (EC: 1.2.1.10) adhA alcohol dehydrogenase (EC: 1.1.1.1) L. lactis ypjA dehydrogenase L. lactis eutG bifunctional acetaldehyde-CoA/alcohol dehydrogenase M. succiniciproducens adhC AdhC protein M. succiniciproducens adhB alcohol dehydrogenase II (EC: 1.1.1.1) P. fluorescens adh alcohol dehydrogenase, zinc-containing (EC: 1.1.1.1) P. fluorescens adhC alcohol dehydrogenase class III P. fluorescens adhA alcohol dehydrogenase P. putida FDH1 Glutathione-dependent formaldehyde dehydrogenase (FDH) P. stipitis (FALDH) (FLD) SAD1 secondary alcohol dehydrogenase (SADH1) P. stipitis SAD2 secondary alcohol dehydrogenase (SADH2) P. stipitis ADH1 alcohol dehydrogenase P. stipitis IFR1 Zinc-binding oxidoreductase alcohol dehydrogenase P. stipitis (EC: 1.1.1.1 1.6.5.5) ADH2 alcohol dehydrogenase P. stipitis ypch00392 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli ypf00190 putative alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli adhA2 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli ypch01140 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli ypb00013 iron alcohol dehydrogenase protein R. etli ypch00170 zinc-binding oxidoreductase protein R. etli adhA1 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli adhCch formaldehyde dehydrogenase (glutathione)/alcohol R. etli dehydrogenase protein (EC: 1.1.1.1 1.1.1.284) ypch00995 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli ypb00024 putative alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli ypf00099 Zn-dependent oxidoreductase protein (EC: 1.1.1.1) R. etli ypch00832 alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli adhE alcohol dehydrogenase protein (EC: 1.1.1.1) R. etli adhCf formaldehyde dehydrogenase (glutathione)/alcohol R. etli dehydrogenase protein (EC: 1.1.1.1 1.1.1.284) adh alcohol dehydrogenase (EC: 1.1.1.1) R. eutropha rplK 50S ribosomal protein L11 (EC: 1.1.1.1) R. eutropha adhC alcohol dehydrogenase, class III (EC: 1.1.1.284) R. eutropha adhP alcohol dehydrogenase, Zn-dependent (EC: 1.1.1.1) R. eutropha adh alcohol dehydrogenase (EC: 1.1.1.1) R. opacus adhP alcohol dehydrogenase (EC: 1.1.1.1) S. aureus adhA alcohol dehydrogenase S. aureus adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. aureus (EC: 1.1.1.1) adh1 alcohol dehydrogenase S. aureus adhC alcohol dehydrogenase (EC: 1.1.1.1) S. aureus adhA2 alcohol dehydrogenase S. avermitilis pteB dehydrogenase S. avermitilis adhA1 alcohol dehydrogenase S. avermitilis adhA6 alcohol dehydrogenase S. avermitilis adhA3 alcohol dehydrogenase S. avermitilis ADH4 Adh4p (EC: 1.1.1.190 1.1.1.1) S. cerevisiae ADH2 Adh2p (EC: 1.1.1.190 1.1.1.1) S. cerevisiae ADH3 Adh3p (EC: 1.1.1.190 1.1.1.1) S. cerevisiae ADH5 Adh5p (EC: 1.1.1.190 1.1.1.1) S. cerevisiae SFA1 Bifunctional enzyme containing both alcohol dehydrogenase S. cerevisiae and glutathione-dependent formaldehyde dehydrogenase activities, functions in formaldehyde detoxification and formation of long chain and complex alcohols, regulated by Hog1p-Sko1p (EC: 1.1.1.284 1.1.1.1) ADH1 Adh1p (EC: 1.1.1.190 1.1.1.1) S. cerevisiae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. enterica (EC: 1.1.1.1 1.2.1.10) adh2 putative iron-containing alcohol dehydrogenase S. enterica adh3 alcohol dehydrogenase class III S. enterica adhP alcohol dehydrogenase S. enterica adhA alcohol dehydrogenase S. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. pneumoniae (EC: 1.1.1.1) adhP alcohol dehydrogenase (EC: 1.1.1.1) S. pneumoniae adhB alcohol dehydrogenase, zinc-containing S. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase Y. pestis (EC: 1.1.1.1) adhC alcohol dehydrogenase Y. pestis

According to the present invention, the alcohol dehydrogenase enzyme has specific activity for prenal or 3-methyl-2-butenal (number CAS 107-86-8), an aldehyde of formula:

and converts it into prenol with the hydrogen donor NADH. This enzyme is also named 3-methyl-2-butenal dehydrogenase in the invention.

An aldehyde dehydrogenase (CoA-acylating) enzyme designates an enzyme catalyzing the following reaction, in one way or in the other: an acyl-CoA+NAD(P)H+H+

an aldehyde+NAD++Coenzyme A

Other known names are: aldehyde:NAD+ oxidoreductase (CoA-acylating), aldehyde dehydrogenase (acylating); acylating aldehyde dehydrogenase; Coenzyme A-acylating aldehyde dehydrogenase; aldehyde dehydrogenase (acetylating); aldehyde:NAD(P)+ oxidoreductase (CoA-acetylating); the common abbreviation is ALDH.

This enzyme always functions with an ‘acceptor’ of hydrogen, such as NAD+, NADP+, or other specific acceptors. The activity of this enzyme is the conversion of an acyl-CoA into an aldehyde, and/or the conversion of an aldehyde into an acyl-CoA with a donor of hydrogen.

In a specific aspect of the invention, the enzyme having aldehyde dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 2:

Gene name Enzyme name Organism eutE acetaldehyde dehydrogenase (acetylating) (EC: 1.2.1.10) A. hydrophila mhpF acetaldehyde dehydrogenase (EC: 1.2.1.10) B. megaterium eutE acetaldehyde dehydrogenase (EC: 1.2.1.10) B. megaterium adhE2 bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. acetobutylicum adhE1 bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. acetobutylicum adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. difficile (EC: 1.2.1.10 1.1.1.1) aad bifunctional acetaldehyde-CoA/alcohol dehydrogenase C. kluyveri (EC: 1.1.1.—1.2.1.—) mhpF acetaldehyde-CoA dehydrogenase II, NAD-binding E. coli (EC: 1.2.1.10) adhE fused acetaldehyde-CoA dehydrogenase/iron-dependent E. coli alcohol dehydrogenase/pyruvate-formate lyase deactivase (EC: 1.2.1.10 1.1.1.1) adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase E. faecalis mhpF acetaldehyde dehydrogenase K. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase K. pneumoniae adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase L. lactis (EC: 1.2.1.10) eutG bifunctional acetaldehyde-CoA/alcohol dehydrogenase M. succiniciproducens mhpF acetaldehyde dehydrogenase (EC: 1.2.1.10) R. eutropha cmtH acetaldehyde dehydrogenase (EC: 1.2.1.10) R. opacus adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. aureus (EC: 1.1.1.1) adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. enterica (EC: 1.1.1.1 1.2.1.10) adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase S. pneumoniae (EC: 1.1.1.1) adhE metal binding site; other site (EC: 1.2.1.10) S. typhimurium adhE bifunctional acetaldehyde-CoA/alcohol dehydrogenase Y. pestis (EC: 1.1.1.1)

According to the present invention, the aldehyde dehydrogenase enzyme has specific activity for 3-methylcrotonyl-CoA, an acyl-CoA of formula:

and converts it into prenal with the hydrogen donor NADH. In the invention, this enzyme is also named 3-methyl-2-butanol dehydrogenase.

In a preferred embodiment of the invention, the alcohol dehydrogenase and aldehyde dehydrogenase activities are catalyzed by the same enzyme, capable of both functions; in a specific embodiment, said enzyme capable of both functions is the alcohol-aldehyde dehydrogenase enzyme (AdhE enzyme).

Examples of enzymes possessing both activities are enzymes previously listed called “bifunctional acetaldehyde-CoA/alcohol dehydrogenase”.

Preferentially, the recombinant microorganism expresses an AdhE enzyme that is heterologous to the microorganism.

According to a specific embodiment, the AdhE enzyme has specificity for the substrate 3-methylcrotonyl-CoA.

The term “specificity” designates affinity of an enzyme for a precise substrate. According to this invention specificity of AdhE enzyme means that this enzyme recognizes the 3-methylcrotonyl-CoA as preferred substrate among all other substrates.

According to a preferred embodiment of the invention, the AdhE enzyme is AdhE1 from Clostridium acetobutylicum.

According to a more preferred embodiment of the invention, the AdhE enzyme is encoded by the gene adhE2 from Clostridium acetobutylicum (listed in table 1 and table 2).

Prenol Bisosynthesis by Leucine Pathway

In this aspect of the invention, the biosynthesis pathway of 3-methylcrotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 4-methyl-2-oxopentanoate and 3-methylbutanoyl-CoA.

The whole pathway for the biosynthesis of prenol according to this embodiment of the invention is illustrated in FIG. 1, entitled “Metabolic pathway for biosynthesis of prenol, from the leucine pathway”.

The first reaction of the conversion of 4-methyl-2-oxopentanoate into 3-methylbutanoyl-CoA is catalysed by the branched-chain keto-acid dehydrogenase complex. This complex is composed of four subunits E1α, E1β, E2 and E3. This enzymatic complex has been identified in several species, and in particular in:

-   -   Bacillus substilis (genes bkdAA, bkdAB, bkdB, lpdV), for         reference: Perham and Lowe, (1988).     -   Pseudomonas putida (genes bkdA1, bkdA2, bkdB, lpdV): Sykes et         al., (1987).     -   Streptomyces avermitilis, for reference: Skinner et al., (1995).     -   Enterococcus faecalis (operon bkdABCD), for reference: Ward et         al., (1999).     -   Saccharomyces cerevisiae, for reference: Sinclair et al.,         (1993).

In a specific aspect of the invention, the subunit E1 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 3:

Gene name Enzyme name Organism bkdA2 2-oxoisovalerate dehydrogenase beta subunit A. tumefaciens bkdA1 2-oxoisovalerate dehydrogenase alpha subunit A. tumefaciens bkdA2 2-oxoisovalerate dehydrogenase beta subunit (EC: 1.2.4.4) B. brevis bkdA1 2-oxoisovalerate dehydrogenase alpha subunit (EC: 1.2.4.4) B. brevis bfmbAb 3-methyl-2-oxobutanoate dehydrogenase, beta subunit B. cereus (EC: 1.2.4.4) bfmbAa 3-methyl-2-oxobutanoate dehydrogenase, alpha subunit B. cereus (EC: 1.2.4.4) bkdAB 2-oxoisovalerate dehydrogenase E1 component subunit beta B. megaterium (EC: 1.2.4.4) bkdAA 2-oxoisovalerate dehydrogenase E1 component subunit alpha B. megaterium (EC: 1.2.4.4) bkdAA branched-chain alpha-keto acid dehydrogenase E1 subunit B. subtilis (EC: 1.2.4.4) bkdAB branched-chain alpha-keto acid dehydrogenase E1 subunit B. subtilis (EC: 1.2.4.4) bkdB branched-chain alpha-keto acid dehydrogenase, E1 component, E. faecalis beta subunit bkdA branched-chain alpha-keto acid dehydrogenase, E1 component, E. faecalis alpha subunit BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptide H. sapiens (EC: 1.2.4.4) BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide H. sapiens (EC: 1.2.4.4) bkdB TPP-dependent branched-chain alpha-keto acid L. casei dehydrogenase, E1 beta subunit (branched-chain alpha-keto acid dehydrogenase, E1 component, beta subunit) bkdA branched-chain alpha-keto acid dehydrogenase, E1 component, L. casei alpha subunit Bckdha branched chain ketoacid dehydrogenase E1, alpha polypeptide M. musculus (EC: 1.2.4.4) Bckdhb branched chain ketoacid dehydrogenase E1, beta polypeptide M. musculus (EC: 1.2.4.4) bkdA1 2-oxoisovalerate dehydrogenase (alpha subunit) P. aeruginosa bkdA2 2-oxoisovalerate dehydrogenase (beta subunit) P. aeruginosa bkdA2 2-oxoisovalerate dehydrogenase, beta subunit P. putida bkdA1 3-methyl-2-oxobutanoate dehydrogenase P. putida bkDa1 2-oxoisovalerate dehydrogenase alpha subunit protein R. etli bkDa2 2-oxoisovalerate dehydrogenase beta subunit protein R. etli Bckdhb branched chain keto acid dehydrogenase E1, beta polypeptide R. norvegicus (EC: 1.2.4.4) Bckdha branched chain ketoacid dehydrogenase E1, alpha polypeptide R. norvegicus (EC: 1.2.4.4) bfmBAA 2-oxoisovalerate dehydrogenase alpha subunit (EC: 1.2.4.4) S. aureus bfmBAB 2-oxoisovalerate dehydrogenase beta subunit (EC: 1.2.4.4) S. aureus

In a specific aspect of the invention, the subunit E2 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 4:

Gene name Enzyme name Organism bkdB branched-chain alpha-keto acid dehydrogenase subunit E2 A. tumefaciens bkdB lipoamide acyltransferase component of branched-chain B. brevis alpha-keto acid dehydrogenase complex (EC: 2.3.1.168) bfmbB branched-chain alpha-keto acid dehydrogenase subunit E2 B. cereus (EC: 2.3.1.—) bkdB branched-chain alpha-keto acid dehydrogenase complex B. megaterium lipoamide acyltransferase E2 component (EC: 2.3.1.168) bkdB branched-chain alpha-keto acid dehydrogenase subunit E2 B. subtilis (EC: 2.3.1.—) bkdC branched-chain alpha-keto acid, E2 component, E. faecalis dihydrolipoamide acetyltransferase DBT dihydrolipoamide branched chain transacylase E2 H. sapiens (EC: 2.3.1.168) bkdC branched-chain alpha-keto acid, E2 component, L. casei dihydrolipoamide acetyltransferase Dbt dihydrolipoamide branched chain transacylase E2 M. musculus (EC: 2.3.1.168) bkdB branched-chain alpha-keto acid dehydrogenase subunit E2 P. aeruginosa bkdB branched-chain alpha-keto acid dehydrogenase subunit E2 P. putida bkdB branched-chain alpha-keto acid dehydrogenase subunit E2 R. etli (EC: 2.3.1.12) Dbt dihydrolipoamide branched chain transacylase E2 R. norvegicus (EC: 2.3.1.168) bfmB lipoamide acyltransferase component of branched-chain S. aureus alpha-keto acid dehydrogenase complex (EC: 2.3.1.—)

In a specific aspect of the invention, the subunit E3 of the enzymatic complex having branched-chain keto acid dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 5:

Gene name Enzyme name Organism lpdA dihydrolipoamide dehydrogenase (EC: 1.8.1.4) A. tumefaciens pdhD dihydrolipoyl dehydrogenase (EC: 1.8.1.4) B. brevis acoL dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. brevis lpd dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. brevis acoL dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. cereus pdhD pyruvate dehydrogenase complex E3 component, B. megaterium dihydrolipoamide dehydrogenase (EC: 1.8.1.4) lpdV branched-chain alpha-keto acid dehydrogenase B. megaterium complex dihydrolipoamide dehydrogenase (EC: 1.8.1.4) acoL acetoin dehydrogenase E3 component B. megaterium (dihydrolipoamide dehydrogenase) (EC: 1.8.1.4) pdhD dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. subtilis acoL dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. subtilis lpdV dihydrolipoamide dehydrogenase (EC: 1.8.1.4) B. subtilis bkdD branched-chain alpha-keto acid dehydrogenase, E3 E. faecalis component, dihydrolipoamide dehydrogenase lpdA dihydrolipoamide dehydrogenase (EC: 1.8.1.4) E. faecalis DLD dihydrolipoamide dehydrogenase (EC: 1.8.1.4) H. sapiens bkdD dihydrolipoyl dehydrogenase (EC: 1.8.1.4) L. casei pdhD dihydrolipoamide dehydrogenase (EC: 1.8.1.4) L. casei Dld dihydrolipoamide dehydrogenase (EC: 1.8.1.4) M. musculus lpd3 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. aeruginosa lpdG dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. aeruginosa lpdV dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. aeruginosa lpdG dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. putida lpd3 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. putida lpdV dihydrolipoamide dehydrogenase (EC: 1.8.1.4) P. putida lpdAc dihydrolipoamide dehydrogenase (EC: 1.8.1.4) R. etli lpdAch1 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) R. etli lpdAch2 dihydrolipoamide dehydrogenase (EC: 1.8.1.4) R. etli pdhD dihydrolipoamide dehydrogenase (EC: 1.8.1.4) S. aureus lpdA dihydrolipoamide dehydrogenase (EC: 1.8.1.4) S. aureus

Preferentially, the enzymatic complex having branched-chain keto acid dehydrogenase activity is the complex from P. putida.

The second reaction of the conversion of 3-methylbutanoyl-CoA into 3-methylcrotonyl-CoA is catalysed by an acyl-CoA dehydrogenase, in particular an isovaleryl-CoA dehydrogenase. This enzyme has been identified in several species, and in particular in:

-   -   Pseudomonas aeruginosa (gene liuA), for reference:         Förster-Fromme and Jendrossek (2008).     -   Streptomyces coelicolor and Streptomyces avermitilis (acdH), for         reference: Zhang et al., (1999).     -   Caenorhabditis elegans (ivd) Mohsen et al., (2001).     -   Arabidopsis thaliana (ivd): Däschner et al., (2001).

In a specific aspect of the invention, the enzyme having acyl-CoA dehydrogenase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 6:

Gene name Enzyme name Organism IBR3 IBR3 (IBA-RESPONSE 3); acyl-CoA dehydrogenase/ A. thaliana oxidoreductase (EC: 1.3.99.3) IVD IVD (ISOVALERYL-CoA-DEHYDROGENASE); ATP A. thaliana binding/isovaleryl-CoA dehydrogenase (EC: 1.3.99.12) acd acyl-CoA dehydrogenase A. tumefaciens acd acyl-CoA dehydrogenase A. tumefaciens acdA acyl-CoA dehydrogenase, short-chain specific B. cereus (EC: 1.3.99.3) bcd acyl-CoA dehydrogenase (EC: 1.3.99.3) B. cereus acdA acyl-CoA dehydrogenase (EC: 1.3.99.3) B. megaterium fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) B. megaterium mmgC acyl-CoA dehydrogenase (EC: 1.3.99.3) B. megaterium acdh-10 Acyl CoA DeHydrogenase C. elegans acdh-7 Acyl CoA DeHydrogenase C. elegans acdh-8 Acyl CoA DeHydrogenase C. elegans ivd-1 IsoValeryl-CoA Dehydrogenase C. elegans fadE acyl coenzyme A dehydrogenase (EC: 1.3.99.3) E. coli ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain H. sapiens (EC: 1.3.99.3) IVD isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) H. sapiens fadE1 acyl-CoA dehydrogenase FadE1 (EC: 1.3.99.—) M. bovis fadE13 acyl-CoA dehydrogenase FadE13 (EC: 1.3.99.—) M. bovis fadE2 acyl-CoA dehydrogenase FadE2 (EC: 1.3.99.—) M. bovis fadE20 acyl-CoA dehydrogenase FadE20 (EC: 1.3.99.—) M. bovis fadE23 acyl-CoA dehydrogenase FadE23 M. bovis fadE4 acyl-CoA dehydrogenase FadE4 (EC: 1.3.99.—) M. bovis ibd2 isobutyryl-CoA dehydrogenase (EC: 1.3.99.10) M. extorquens Acadm acyl-Coenzyme A dehydrogenase, medium chain M. musculus (EC: 1.3.99.3) Ivd isovaleryl coenzyme A dehydrogenase (EC: 1.3.99.10) M. musculus fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) P. aeruginosa liuA putative isovaleryl-CoA dehydrogenase P. aeruginosa acd-6 acyl-CoA dehydrogenase family protein P. fluorescens fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) P. fluorescens fadE1 acyl-CoA dehydrogenase family protein P. fluorescens fadE13 hypothetical protein P. fluorescens fadE20 acyl-CoA dehydrogenase P. fluorescens ivd isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) P. fluorescens fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) P. putida ivd acyl-CoA dehydrogenase domain containing protein P. putida acd1 acyl-CoA dehydrogenase protein R. etli acd2 acyl-CoA dehydrogenase protein R. etli ivdH isovaleryl-CoA dehydrogenase protein R. etli abmD putative acyl-CoA dehydrogenase (EC: 1.3.99.—) R. eutropha acaD acyl-CoA dehydrogenase (EC: 1.3.99.3) R. eutropha aidB acyl-CoA dehydrogenase, short-chain specific (AidB R. eutropha protein) (EC: 1.3.99.3) ivd1 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) R. eutropha ivd2 isovaleryl-CoA dehydrogenase (EC: 1.3.99.10) R. eutropha fadE15 acyl-CoA dehydrogenase S. avermitilis fadE17 acyl-CoA dehydrogenase S. avermitilis fadE7 acyl-CoA dehydrogenase S. avermitilis acdH acyl-CoA dehydrogenase S. avermitilis acdH2 acyl-CoA dehydrogenase S. coelicolor acdH3 acyl-CoA dehydrogenase S. coelicolor acdC putative acyl-CoA dehydrogenase S. enterica fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) S. enterica acdA acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea acdA-3 acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea fadE1 acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea fadE13 acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea fadE21 putative acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea fadE22 putative acyl-CoA dehydrogenase S. erythraea fadE31 putative acyl-CoA dehydrogenase (EC: 1.3.99.3) S. erythraea fadE domain of unknown function (DUF1974); region: S. typhimurium DUF1974; pfam09317 (EC: 1.3.99.3) acdA acyl-CoA dehydrogenase X. oryzae fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) X. oryzae fadE acyl-CoA dehydrogenase (EC: 1.3.99.3) Y. pestis

In a preferred embodiment of the invention, the acyl-CoA dehydrogenase is encoded by the gene acdH from S. avermitilis.

In a specific embodiment of the invention, in the recombinant microorganism, at least one of the following enzymes is overexpressed: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase.

In a more specific aspect of the invention, in the recombinant microorganism, branched chain keto acid dehydrogenase complex and the enzyme acyl-CoA dehydrogenase, that are overexpressed in the genetically modified microorganism, are heterologous.

In particular, the branched chain keto acid dehydrogenase is encoded by one or several genes issued from Bacillus subtilis (bkdAA, bkdAB, bkdB, lpdV), Pseudomonas putida (bkdA1, bkdA2, bkdB, lpdV), Streptomyces avermitilis, Enterococcus faecalis (bkdABCD) or Saccharomyces cerevisiae.

In particular the acyl-CoA dehydrogenase is encoded by a gene from Pseudomonas aeruginosa (liuA), Streptomyces coelicolor, Streptomyces avermitilis (acdH), Caenorhabditis elegans (ivd) or Arabidopsis thaliana (ivd).

In another aspect of the invention, the microorganism is further modified to improve the availability of at least one biosynthesis intermediate chosen among pyruvate, 3-methyl-2-oxobutanoic acid and 4-methyl-2-oxopentanoate.

In order to optimize pyruvate availability, the microorganism is modified to overexpress at least one gene involved in pyruvate biosynthesis pathway, chosen among gene coding for phosphoglycerate mutase (gpmA and pgmI in E. coli or homologous gene), enolase (eno in E. coli or homologous gene) or pyruvate kinase (pykA and pykF in E. coli or homologous gene). Alternatively or in combination, at least one gene involved in pyruvate degradation pathway is attenuated. This gene is chosen among pyruvate oxidase (poxB in E. coli or homologous gene), phosphate acetyltransferase (pta in E. coli or homologous gene), acetate kinase (ackA in E. coli or homologous gene), aldehyde/alcohol dehydrogenase (adhE in E. coli or homologous gene), pyruvate dehydrogenase operon repressor (pdhR in E. coli or homologous gene) or lactate dehydrogenase (pfl, lldD, ldhA or did in E. coli or homologous gene).

In order to optimize 3-methyl-2-oxobutanoic acid availability, the microorganism is modified to overexpress at least one gene involved in the L-valine biosynthesis pathway, chosen among gene coding for acetolactate synthase (ilvl, ilvH, ilvN and ilvB in E. coli or homologous genes), keto-acid reductoisomerase (ilvC in E. coli or homologous gene) and dihydroxy-acid dehydratase (ilvD in E. coli or homologous gene). Alternatively or in combination, the gene encoding the branched chain amino acid transaminase (ilvE in E. coli or homologous gene) is deleted in the microorganism of the invention. In another embodiment of the invention, the ilvN gene is modified so as to produce an IlvN protein which is feedback deregulated. Such mutations of ilvN are disclosed in Park et al., 2011.

In order to optimize 4-methyl-2-oxopentanoate availability, the microorganism is modified to overexpress at least one gene chosen among the genes coding for 3-isopropylmalate dehydratase (leuC, leuD in E. coli or homologous genes), 3-isopropylmalate dehydrogenase (leuB in E. coli or homologous gene) or 2-isopropylmalate synthase (leuA in E. coli or homologous gene). Alternatively or in combination, the gene encoding the branched chain amino acid transaminase (ilvE in E. coli or homologous gene) is deleted in the microorganism of the invention. In another embodiment of the invention, the leuA gene is modified so as to produce a LeuA protein which is feedback deregulated. Such mutations of leuA are disclosed in patent application U.S. Pat. No. 6,403,342.

Prenol Bisosynthesis by HMG-CoA Pathway

In this aspect of the invention, the biosynthesis pathway of 3-methylcrotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) and 3-methylglutaconyl-CoA.

The whole pathway for the biosynthesis of prenol according to this embodiment of the invention is illustrated in FIG. 2, entitled “Metabolic pathway for biosynthesis of prenol, from an intermediate of the HMG-CoA pathway”.

The first reaction of condensation of two acetyl-CoA molecules into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) is catalysed successively by two enzymes: (1) an acetyl-CoA acetyltransferase, and (2) a 3-hydroxy-3-methylglutaryl-CoA synthase.

Genes coding for enzymes having an acetyl-CoA acetyltransferase activity have been identified in several species, and in particular in Escherichia coli (gene atoB), Clostridium acetobutylicum (thlA) and Saccharomyces cerevisiae (ERG10).

In a specific aspect of the invention, the enzyme having acetyl-CoA acetyltransferase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 7:

Gene name Enzyme name Organism ACAT2 ACAT2 (ACETOACETYL-CoA THIOLASE 2); acetyl- A. thaliana CoA C-acetyltransferase/catalytic (EC: 2.3.1.9) mmgA acetyl-CoA acetyltransferase (EC: 2.3.1.9) B. brevis thl acetyl-CoA acetyltransferase B. cereus atoB acetyl-CoA acetyltransferase B. cereus thiL acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. acetobutylicum ERG10 similar to Acetoacetyl-CoA Thiolase A; mevalonate/sterol C. albicans pathway thlA1 acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. difficile thlA2 putative acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. difficile kat-1 3-Ketoacyl-CoA Thiolase C. elegans thlA1 acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. kluyveri thlA2 acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. kluyveri thlA3 acetyl-CoA acetyltransferase (EC: 2.3.1.9) C. kluyveri atoB acetyl-CoA acetyltransferase (EC: 2.3.1.9) E. coli yqeF predicted acyltransferase E. coli ACAT1 acetyl-CoA acetyltransferase 1 (EC: 2.3.1.9) H. sapiens ACAT2 acetyl-CoA acetyltransferase 2 (EC: 2.3.1.9) H. sapiens yqeF acetyl-CoA acetyltransferase K. pneumoniae thiL acetyl coenzyme A acetyltransferase (EC: 2.3.1.9) L. lactis fadA acetyl coenzyme A acetyltransferase (EC: 2.3.1.9) L. lactis fadA acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis fadA6 acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis fadA3 acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis fadA4 acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis fadA2 acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis fadA5 acetyl-CoA acetyltransferase (EC: 2.3.1.9) M. bovis phaA beta-ketothiolase (EC: 2.3.1.16) M. dichloromethanicum Acat1 acetyl-Coenzyme A acetyltransferase 1 (EC: 2.3.1.9) M. musculus Acat2 acetyl-Coenzyme A acetyltransferase 2 (EC: 2.3.1.9) M. musculus atoB acetyl-CoA acetyltransferase (EC: 2.3.1.9) P. aeruginosa atoB acetyl-CoA acetyltransferase P. putida fadAx acetyl-CoA acetyltransferase P. putida ERG10 acetyl-CoA acetyltransferase (EC: 2.3.1.9) P. stipitis fadA acetyl-CoA acetyltransferase (EC: 2.3.1.9) R. etli phbAch acetyl-CoA acetyltransferase (EC: 2.3.1.9) R. etli phbAf acetyl-CoA acetyltransferase (beta-ketothiolase) protein R. etli (EC: 2.3.1.9) phaA acetyl-CoA acetyltransferase (EC: 2.3.1.9) R. eutropha bktB beta-ketothiolase (EC: 2.3.1.9) R. eutropha fadA acetyl-CoA acetyltransferase (EC: 2.3.1.16) R. opacus pcaF acetyl-CoA acetyltransferase (EC: 2.3.1.174) R. opacus fadA5 3-ketoacyl-CoA thiolase/acetyl-CoA acetyltransferase S. avermitilis fadA1 acetyl-CoA acetyltransferase (EC: 2.3.1.9) S. avermitilis fadA7 acetyl-CoA acetyltransferase (EC: 2.3.1.9) S. avermitilis fadA4 acetyl-CoA acetyltransferase (EC: 2.3.1.9) S. avermitilis fadA2 acetyl-CoA acetyltransferase (EC: 2.3.1.9) S. avermitilis fadA3 acetyl-CoA acetyltransferase (EC: 2.3.1.9) S. avermitilis ERG10 Acetyl-CoA C-acetyltransferase (acetoacetyl-CoA S. cerevisiae thiolase), cytosolic enzyme that transfers an acetyl group from one acetyl-CoA molecule to another, forming acetoacetyl-CoA; involved in the first step in mevalonate biosynthesis (EC: 2.3.1.9) atoB acetoacetyl-CoA thiolase X. oryzae yfcY acetyl-CoA acetyltransferase (EC: 2.3.1.9) X. oryzae fadA acetyl-CoA acetyltransferase (EC: 2.3.1.9) X. oryzae acat1 acetyl-Coenzyme A acetyltransferase 1 (EC: 2.3.1.9) X. tropicalis acat2 acetyl-Coenzyme A acetyltransferase 2 (EC: 2.3.1.9) X. tropicalis

In a preferred embodiment of the invention, the acetyl-CoA acetyltransferase is encoded by the gene atoB from E. coli.

Genes coding for enzymes having a 3-hydroxy-3-methylglutaryl-CoA synthase activity have been identified in several species, and in particular in Enterococcus faecalis (gene mvaS), and Saccharomyces cerevisiae (ERG13).

In a specific aspect of the invention, the enzyme having 3-hydroxy-3-methylglutaryl-CoA synthase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 8:

Gene name Enzyme name Organism MVA1 MVA1; acetyl-CoA C-acetyltransferase/ A. thaliana hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) pksG acetyl-S-AcpK beta-ketothioester polyketide intermediate B. subtilis transferase HMGCS1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 B. taurus (soluble) HMGCS2 3-hydroxy-3-methylglutaryl-CoA synthase 2 B. taurus (mitochondrial) (EC: 2.3.3.10) ERG13 3-hydroxy-3-methylglutaryl coenzyme A synthase C. albicans HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble) E. caballus mvaS 3-hydroxy-3-methylglutaryl-CoA synthase E. faecalis HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1 (soluble) H. sapiens (EC: 2.3.3.10) HMGCS2 3-hydroxy-3-methylglutaryl-CoA synthase 2 H. sapiens (mitochondrial) (EC: 2.3.3.10) hmcM hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) L. lactis Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 M. musculus (EC: 2.3.3.10) Hmgcs2 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 M. musculus (EC: 2.3.3.10) mvaS 3-hydroxy-3-methylglutaryl coenzyme A synthase S. aureus ERG13 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, S. cerevisiae catalyzes the formation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA; involved in the second step in mevalonate biosynthesis (EC: 2.3.3.10) 3-hydroxy-3-methylglutaryl-Coenzyme A synthase S. avermitilis pksG hydroxymethylglutaryl-CoA synthase (EC: 2.3.3.10) S. erythraea hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 X. tropicalis (soluble) (EC: 2.3.3.10) pksG putative hydroxymethylglutaryl-coenzyme A synthase Y. pestis

Preferentially the 3-hydroxy-3-methylglutaryl-CoA synthase is encoded by the gene mvaS from Enterococcus faecalis.

The second reaction of the conversion of HMG-CoA into 3-methylcrotonyl-CoA is catalysed successively by two enzymes: (1) 3-methylglutaconyl-CoA hydratase, and (2) 3-methylglutaconyl-CoA decarboxylase.

Enzymes having a 3-methylglutaconyl-CoA hydratase activity have been identified in several species and in particular in Galactomyces reessi (See for reference: Dhar et al., 2002).

In a specific aspect of the invention, the enzyme having 3-methylglutaconyl-CoA hydratase activity is encoded by a gene chosen among a list of genes well known in the art, including but not limited to the genes listed in table 9:

Gene name Enzyme name Organism mgh 3-methylglutaconyl-CoA hydratase (EC: 4.2.1.18) A. baumannii fadB1 enoyl-CoA hydratase A. radiobacter paaG enoyl-CoA hydratase (EC: 4.2.1.17) B. multivorans AUH AU RNA binding protein/enoyl-CoA hydratase (EC: 4.2.1.18) B. taurus AUH AU RNA binding protein/enoyl-CoA hydratase C. familiaris paaF methylglutaconyl-CoA hydratase (EC: 4.2.1.18) C. hutchinsonii auh AU RNA binding protein/enoyl-Coenzyme A hydratase D. rerio (EC: 4.2.1.18) mvaA enoyl-CoA hydratase (EC: 4.2.1.18) D. shibae 3-methylglutaconyl-CoA hydratase G. reessi AUH AU RNA binding protein/enoyl-CoA hydratase (EC: 4.2.1.18) H. sapiens caiD enoyl-CoA hydratase/carnithine racemase (EC: 4.2.1.17) H. seropedicae AUH AU RNA binding protein/enoyl-CoA hydratase M. mulatta Auh AU RNA binding protein/enoyl-coenzyme A hydratase M. musculus (EC: 4.2.1.18) MXAN_3757 methylglutaconyl-CoA hydratase M. xanthus liuC gamma-carboxygeranoyl-CoA hydratase P. aeruginosa gnyH gamma-carboxygeranoyl-CoA hydratase P. aeruginosa echA7 gamma-carboxygeranoyl-CoA hydratase P. fluorescens AUH AU RNA binding protein/enoyl-CoA hydratase P. troglodytes menB naphthoate synthase (EC: 4.1.3.36) R. capsulatus Auh AU RNA binding protein/enoyl-coenzyme A hydratase R. norvegicus (EC: 4.2.1.18) eccH2 enoyl-CoA hydratase (EC: 4.2.1.17) S. meliloti fadB probable enoyl-CoA hydratase (EC: 4.2.1.17) S. ruber auh AU RNA binding protein/enoyl-Coenzyme A hydratase X. laevis (EC: 4.2.1.18) auh AU RNA binding protein/enoyl-Coenzyme A hydratase X. tropicalis (EC: 4.2.1.18)

Preferentially the 3-methylglutaconyl-CoA hydratase is encoded by the gene MXAN_3757 from Myxococcus xanthus.

At least one gene coding for an enzyme having a 3-methylglutaconyl-CoA decarboxylase activity has been identified in Myxococcus xanthus (See for reference: Bode et al., 2009).

Preferentially the 3-methylglutaconyl-CoA decarboxylase is encoded by the genes MXAN_4264 and MXAN_4265 from Myxococcus xanthus, each of them coding for one subunit of the enzyme.

In a specific embodiment of the invention, in the recombinant microorganism, at least one of the following enzymes is overexpressed: an acetyl-CoA acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, and a 3-methylglutaconyl-CoA decarboxylase.

In another aspect of the invention, the microorganism is further modified to improve the availability of acetyl-CoA by:

-   -   overexpressing at least one gene encoding the pyruvate         dehydrogenase chosen among aceE, aceF or lpd. In a specific         aspect of the invention, inactivation of pdhR coding for a         repressor leads to an overexpression of aceE and aceF.     -   modifying the lpd gene so as to produce a mutant, feedback         deregulated Lpd enzyme. Such mutations of lpd are disclosed in         patent application WO2005073364.     -   attenuating at least one gene chosen among aldehyde/alcohol         dehydrogenase (adhE), phosphate acetyltransferase (pta), acetate         kinase (ackA) or citrate Synthase (gltA).

Preferentially, the microorganism of the invention is selected among Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, Corynebacteriaceae and Saccharomycetaceae. More preferentially the microorganism is a species of Escherichia, Clostridium, Bacillus, Klebsiella, Pantoea, Salmonella, Pseudomonas, Corynebacterium or Saccharomyces.

According to a specific aspect of the invention, the microorganism is from the species Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, Saccharomyces cerevisiae, Corynebacterium glutamicum or Bacillus subtilis.

An especially preferred simple carbon source is glucose. Another preferred simple carbon source is sucrose.

In some embodiments of the invention, the culture medium comprises a carbon source being a by-product of another process using biomass as starting material, or eventually, the product of mechanical and/or chemical and/or enzymatic, and in such instance in vitro or in vivo, degradation of biomass, such as degradation of cellulose.

According to a specific aspect of the invention, the fermentative production of prenol comprises a step of isolation of the prenol from the culture medium. Recovering the prenol from the culture medium is a routine task for a man skilled in the art. It may be achieved by a number of techniques well known in the art including but not limiting to distillation, gas-stripping, pervaporation or liquid extraction. The expert in the field knows how adapt parameters of each technic dependant of the characteristics of the material to be separated.

Distillation may involve an optional component different from the culture medium in order to facilitate the isolation of prenol by forming azeotrope and notably with water. This optional component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, trichloroethylene, octane, diethylether or a mixture thereof.

Gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen or mixture thereof.

Liquid extraction is achieved with organic solvent as the hydrophobe phase such as pentane, hexane, heptane, dodecane.

Conversion of Prenol into Isoprene:

In a specific embodiment, the invention is also related to a method for the production of isoprene from prenol. Said method comprises the following successive steps:

-   -   culturing a recombinant microorganism in a culture medium         comprising a source of carbon, wherein in said microorganism,         the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA         as intermediate product, that is converted into prenol by the         action of an alcohol dehydrogenase enzyme and of an aldehyde         dehydrogenase enzyme, and     -   performing a step of chemical dehydratation of the bioproduced         prenol into isoprene.

This conversion of “bioproduced prenol”, i.e. prenol produced from a simple source of carbon by fermentation, into isoprene can be achieved by means and methods known to the man skilled in the art. In particular, this conversion may be achieved by chemical way involving dehydrogenation and dehydration by acid catalyst (chemical dehydratation) such as disclosed in patent application US20100216958.

Another way of conversion is the enzymatic conversion of prenol by action of a prenol kinase, an isopentenyl-diphosphate delta isomerase and an isoprene synthase polypeptide such as disclosed in patent application WO2010031076. These three enzymes may be produced in a different strain than that producing prenol or in the same strain. If they are produced independently from the prenol production, the expressed enzymes or the strains expressing these enzymes can be directly mixed, partly or not, with the fermentation medium of the strain producing prenol or with the culture supernatant wherein prenol is accumulated.

In a specific aspect of the invention, the bioproduced prenol is purified before the step of chemical dehydration.

In another embodiment of the invention, isoprene is purified by standard methods well known in the art. For examples, isoprene can be recovered by gas stripping, extractive distillation with an alcohol such as ethanol, methanol, propanol, or a combination thereof, liquid extraction or solid separation (adsorption, desorption) or combination thereof.

The present invention is also related to a genetically modified microorganism for the fermentative production of prenol such as described above. Specifically, said microorganism overexpresses at least one enzyme chosen among the group consisting of: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase, an acetyl-CoA C-acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, and a 3-methylglutaconyl-CoA decarboxylase. In particular, the genetically modified microorganism comprises a heterologous enzyme AdhE, which has specificity for the substrate 3-methylcrotonyl-CoA.

In said genetically modified microorganism, endogenous sequences may also be knocked out or deleted, to favour the new metabolic pathway for producing prenol.

All techniques for transforming the microorganisms, and regulatory elements used for enhancing the production of prenol, are well known in the art and available in the literature, including the applicant's own patent applications on the modification of biosynthesis pathways in various microorganisms, including WO 2008/052973, WO 2008/052595, WO 2008/040387, WO 2007/144346, WO 2007/141316, WO 2007/077041, WO 2007/017710, WO 2006/082254, WO 2006/082252, WO 2005/111202, WO 2005/073364, WO 2005/047498, WO 2004/076659, the content of which is incorporated herein by reference.

EXAMPLES

The present invention is further defined in the following examples. It should be understood that these example, while indicating preferred embodiments of the invention, are given by way of illustration only. From above disclosure and these examples, the man skilled in the art can make various changes of the invention to adapt it to various uses and conditions without modifying the essentials means of the invention.

In particular, examples show modified Escherichia coli (E. coli) strains, but these modifications can easily be performed in other microorganisms of the same family.

E. coli belongs to the Enterobacteriaceae family, which comprises members that are Gram-negative, rod-shaped, non-spore forming and are typically 1-5 μm in length. Most members have flagella used to move about, but a few genera are non-motile. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. E. coli is one of the most important model organisms, but other important members of the Enterobacteriaceae family include Klebsiella, in particular Klebsiella terrigena, Klebsiella planticola or Klebsiella oxytoca, and Salmonella.

Protocoles

Several protocols are used to construct prenol producing strains and are described in the following examples.

Protocol 1:

Chromosomal modifications by homologous recombination and selection of recombinants (Datsenko and Wanner, 2000)

Allelic replacement or gene disruption in specified chromosomal loci is carried out by homologous recombination as described by Datsenko and Wanner (2000). The chloramphenicol (Cm) resistance cat or the kanamycin (Km) resistance kan flanked by Flp recognition sites, are amplified by PCR by using pKD3 or pKD4 plasmids as template respectively. The resulting PCR products are used to transform the recipient E. coli strain harbouring plasmid pKD46 that expresses the λ, Red (γ, β, exo) recombinase. Antibiotic-resistant transformants are then selected and the thermo-sensitive pKD46 plasmid is removed by cultivating the strain at 42° C. following by an isolation of the strain's culture on LB plates. Single clones are verified for the loss of ampicillin resistance and by PCR analysis with appropriate primers listed in Table 2.

The cat and kan-resistance genes are removed by using plasmid pCP20 as described by Datsenko & Wanner (2000). Antibiotic sensitive clones are then verified by PCR using primers listed in Table 2.

Protocol 2: Transduction of Phage P1

Chromosomal modifications are transferred to a given E. coli recipient strain by P1 transduction. The protocol is composed of 2 steps: (i) preparation of the phage lysate on a donor strain containing the resistance associated chromosomal modification and (ii) infection of the recipient strain by this phage lysate.

Preparation of the Phage Lysate

-   -   Inoculate 100 μl of an overnight culture of the strain MG1655         with the chromosomal modification of interest in 10 ml of LB+Cm         30 μg/ml or Km 50 μg/ml+glucose 0.2%+CaCl₂ 5 mM.     -   Incubate 30 min at 37° C. with shaking.     -   Add 100 μl of P1 phage lysate prepared on the donor strain         MG1655 (approx. 1×10⁹ phage/ml).     -   Shake at 37° C. for 3 hours until the complete lysis of cells.     -   Add 200 μl of chloroform, and vortex.     -   Centrifuge 10 min at 4500 g to eliminate cell debris.     -   Transfer of supernatant to a sterile tube.     -   Store the lysate at 4° C.

Transduction

-   -   Centrifuge 10 min at 1500 g 5 ml of an overnight culture of         the E. coli recipient strain cultivated in LB medium.     -   Suspend the cell pellet in 2.5 ml of MgSO₄ 10 mM, CaCl₂ 5 mM.     -   Infect 100 μl cells with 100 μl P1 phage of strain MG1655 with         the modification on the chromosome (test tube) and as a control         tubes 100 μl cells without P1 phage and 100 μl P1 phage without         cells.     -   Incubate 30 min at 30° C. without shaking.     -   Add 100 μl sodium citrate 1 M in each tube, and vortex.     -   Add 1 ml of LB.     -   Incubate 1 hour at 37° C. with shaking.     -   Centrifuge 3 min at 7000 rpm.     -   Plate on LB+Cm 30 μg/ml or Km 50 μg/ml.

Incubate at 37° C. overnight.

The antibiotic-resistant transductants are then selected and the chromosomal structure of the mutated locus was verified by PCR analysis with appropriates primers listed in Table 2.

TABLE 1 Describe the genotype and corresponding number of intermediate strains and producing strains that appear in the following examples. Strain number Genotype 1 DH5α (pNSTLL-factorXA-adhE2ca) 2 MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20- 22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq- lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07- Ptrc30/RBS01-adhE2ca-TT02) 3 MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) 4 MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/ OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)- TT07)

TABLE 2 Primers used for PCR verifications of chromosomal modifications described in the following examples Location of the  Genes SEQ homology with the name Primers name ID N^(o) chromosomal region Sequences atoB atoB F 17 2323586-2323603 CCATCAATACCGTCAACC atoB R 18 2324536-2324554 CATAAACCTGTCCGTCTCC pdhR pdhR F 21 121197-121217 GAATGTATTTACCCACGGCAG pdhR R 22 123095-123114 GCACGCTCAACACCTTCTTC lpd* Lpd*(A55V) ver F 25 127731-127750 GGTGGTGGTTAGGGTATTAC (A55V) Lpd*(A55V) cer FR 26 129415-129433 CGTGGAGCAAGAAGACTGG

Example 1

Calculation of Maximum Yields for Prenol Production on Glucose and Sucrose

1.1—Parameters Used for Simulations

Simulations were performed with the METEX proprietary software METOPT™. A simplified metabolic network of E. coli was used including a central metabolic network, metabolic pathways for all biomass precursors and specific production pathways as described in FIG. 1 for the leucine pathway and FIG. 2 for the HMG-CoA pathway. A classical biomass composition for E. coli was used. Simulations were performed using either glucose or sucrose carbon source. For sucrose utilization, both the PTS system and the non-PTS system were modelled. As there were no differences on maximal yields calculated, only one yield on sucrose is reported. Maximum prenol yields were calculated from the “leucine pathway” and the “HMG-CoA pathway”. Calculation of a theoretical maximum yield was performed, taking into account no growth and no maintenance. Calculation of a practical maximum yield was performed, taking into account a growth rate of 0.11 h⁻¹ and maintenance energy of 5 mmol_(ATP)·g_(DW) ⁻¹·h⁻¹. All simulations were performed with a specific uptake rate of glucose of 3 mmol·g_(DW) ⁻¹·h⁻¹, or a specific uptake rate of sucrose of 1.5 mmol·g_(DW) ⁻¹·h⁻¹. Simulations were performed under aerobic conditions.

1.2—Simulation Results

Prenol on glucose Prenol on Prenol on Prenol on by the glucose by the sucrose by the sucrose by the “leucine “HMG-CoA “leucine “HMG-CoA pathway” pathway” pathway” pathway” Maximum 0.32 0.32 0.34 0.34 theoretical yield (g/g) Maximum 0.24 0.24 0.26 0.26 practical yield (g/g)

Example 2 Demonstration of the 3-Methyl-2-Butenal Dehydrogenase and 3-Methyl-2-Butenol Dehydrogenase Activities Encoded by the Gene adhE2 of Clostridium acetobutylicum

2.1—Construction of Strain DH5α (pNSTLL-Factor XA-adhE2ca)

pNSTLL-factor XA-adhE2ca plasmid was derived from pSO595 (genbank accession No AY187686) and RBS-Strep-Tag-Linker-factor XA-adhE2ca fragment with the adhE2 gene from Clostridium acetobutylicum ATCC-824 coding for the aldehyde/alcohol dehydrogenase.

In this plasmid, expression of the adhE2ca gene is driven by the promoter of the thiolase gene from Clostridium acetobutylicum. The RBS-Strep-Tag-Linker-factor XA-adhE2ca fragment was amplified by PCR with primers ST_LL_XA_adhE2ca F (SEQ ID No01) and adhE2ca R (SEQ ID No02) using Clostridium acetobutylicum genomic DNA. The PCR product was digested and cloned between the BamHI and SfoI sites of the pSO595. The ligation product was introduced in DH5α strain, the resulting strain DH5α (pNSTLL-factor XA-adhE2ca) is called strain 1 (table 1). The resulting plasmid was verified by DNA sequencing and called: pNSTLL-factor XA-adhE2ca.

ST_LL_XA_adhE2ca F (SEQ ID No 1) TAGGATCC atcaaaatttaggaggttagttagaatg tggtcacatcct caatttgaaaaaggtagtggtggtggtagtggtggtggtagtCCCGGG atcgaagggcgc atgaaagttacaaatcaaaaag

-   -   sequence (bold upper case) for BamHI restriction site and         extrabases     -   sequence (underlined italic lower case) corresponding to RBS         sequence     -   sequence (underlined bold lower case) corresponding to Strep-tag         sequence (Strep-Tag® II, IBA-GmbH)     -   sequence (underlined lower case) corresponding to linker         sequence (artificial sequence)     -   sequence (upper case) for SmaI restriction site     -   sequence (italic lower case) corresponding to factor XA sequence         (Nagai and Thorgersen, 1984)     -   sequence (bold lower case) homologous to the adhE gene of         Clostridium acetobutylicum (36298-36277 (pSOL1), reference         sequence on the NCBI GenBank)

adhE2ca R (SEQ ID No 2) TAAGTGGCGCC TTAAAATGATTTTATATAGATATCC

-   -   sequence (bold upper case) for SfoI restriction site and         extrabases,     -   sequence (italic upper case) homologous to the adhE gene of         Clostridium acetobutylicum (33722-33746 (pSOL1), reference         sequence on the NCBI GenBank)

2.2—Overproduction of the Protein AdhE2ca Protein production was realised in a 1 L Schott bottle. The production strain was inoculated in fifteen precultures of 5 mL LB medium (Sigma 25%) with 2.5 g·L⁻¹ glucose. The temperature was maintained at 37° C. and agitation at 200 RPM.

Theses precultures were used to inoculate an 800 mL culture of MAC medium to an OD_(600 nm) of 0.3. The temperature of the culture was maintained at 37° C. and the agitation at 150 RPM. Ampicilin was added at concentration of 50 mg·L⁻¹ in preculture and culture. When the culture had reached an OD₆₀₀ of 0.8 (approximately 7 hours), the culture was centrifuged and the cell pellet conserved.

TABLE 3 MAC Medium composition. Compound Concentration (g · L⁻¹) Glycerol 20.000 Tryptone 10.000 NaCl 5.000 NaNO₃ 0.085 Yeast extract 5.000 K₂HPO₄ 0.500 FeSO₄•7H₂O 0.050 HEPES 23.000 Nitrilotriacetic acid 0.200 H₂SO₄ (96%) Adjusted to pH 7.3

2.3—Purification of the Protein adhE2

All the purification steps were performed under anaerobic conditions.

Step 1: Preparation of Cell-Free Extracts

About 200 mg of E. coli biomass was suspended in 30 ml of 100 mM Tris HCl, 150 mM NaCl, 1 mM EDTA pH 8 and a protease inhibitor cocktail. The cell suspension was sonicated on ice (Sonics and Materials, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with 30 sec intervals. After sonication, cell debris was removed by centrifugation at 12000 g for 30 min at 4° C. The crude extract was incubated with 0.16 g/L Avidin during 30 min at 4° C. The crude extract was centrifuged at 12000 g for 5 min and filtered through a 0.45 μm filter.

Step 2: Affinity Purification

After Avidin treatment, the crude extract was loaded on a 1 ml StrepTrap HP column (GE Healthcare) equilibrated with 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH8. The column was washed with 10 column volumes of the same buffer. The protein was eluted from the column with 6 column volumes of 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2.5 mM Desthiobiotin pH8. The fractions containing the protein were pooled. For the storage of the protein; the buffer was exchanged using a desalting column (Econo-Pac, Bio-Rad) against 100 mM Hepes pH7.5.

2.4—3-Methyl-2-Butenal Dehydrogenase Assay

3-Methyl-2-butenal dehydrogenase activity was assayed by measuring the initial rate of NADH oxidation under anaerobic conditions with a spectrophotometer at a wavelength of 340 nm and a constant temperature of 30° C. The reaction mixture using 2.5 mM 3-methyl-crotonyl-coA as substrate was carried out in 200 mM HEPES, 144 mM Semicarbazine buffer pH 7.5, 2 mM DTT, 0.2 mM NADH, and about 8 μg of purified enzyme (adhE2) in a final volume of 1 ml. Control assay (blank), lacking the substrate was run in parallel, and the value measured for the control is subtracted to the value measured for the assay in order to take into account the non-specific oxidation of NADH (Epsilon 340 nm=6290 M−1 cm-1).

One unit of enzyme activity was defined as the amount of enzyme that consumed 1 μmol substrate per mM under the conditions of the assay. Specific enzyme activity was expressed as units per mg of protein.

2.5—3-Methyl-2-Butenol Dehydrogenase Assay

3-Methyl-2-butenol dehydrogenase activity was assayed by measuring the initial rate of NADH oxidation under anaerobic conditions with a spectrophotometer at a wavelength of 340 nm and a constant temperature of 30° C. The reaction mixture using 5 mM 3-Methyl-2-butenal as substrate was carried out in 100 mM HEPES buffer pH 7.5, 2 mM DTT, 0.2 mM NADH, and about 12 μg of purified enzyme (adhE2) in a final volume of 1 ml. Control assay (blank), lacking the substrate was run in parallel and the value measured for the control is subtracted to the value measured for the assay in order to take into account non-specific oxidation of NADH (Epsilon 340 nm=6290 M−1 cm-1).

One unit of enzyme activity was defined as the amount of enzyme that consumed 1 μmol substrate per min under the conditions of the assay. Specific enzyme activity was expressed as units per mg of protein.

2.6—Activity of Purified Enzyme AdhE2

Activity of purified enzyme (mUI/mg) 3-Methyl-2-butenal dehydrogenase assay 335 3-Methyl-2-butenol dehydrogenase assay 56

Example 3 Construction of Strain 2 MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02)

3.1—Construction of pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 Plasmid

3.1.1—Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07

Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 is derived from plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C described in patent applications EP11306306.9 and U.S. 61/544,748 to which the ilvD gene from Escherichia coli coding for the dihydroxy-acid dehydratase is added.

In this plasmid, expression of the ilvD gene is driven by a constitutive Ptrc promoter, the ilvD gene is expressed in the operon with ilvBN*(GVM20-22DDF)C and a transcriptional terminator is added downstream of the gene. The ilvD gene is amplified by PCR with primers ilvD F (SEQ ID No03) and ilvD R (SEQ ID No04) using pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C-PilvE-ilvED-TT07 described in patent applications EP11306306.9 and US61/544,748. The PCR product is digested and cloned between the BamHI and NheI sites of the pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)C described in patent applications EP11306306.9 and US61/544,748. The resulting plasmid is verified by DNA sequencing and called: pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07.

ilvD F (SEQ ID No 3) TACTGGCTAGCatacaaaaaatgggacggc

with

-   -   sequence (upper case) for NheI restriction site and extrabases     -   sequence (bold lower case) homologous to the region upstream of         ilvD gene (3951437-3951455, reference sequence in the MySql         relational database (EcoGene.org))

ilvD R (SEQ ID No 4) agcaaggatccGCAGAAAGGCCCACCCGAAGG

with

-   -   sequence (lower case) for BamHI restriction site and extrabases     -   sequence (bold upper case) for T7Te transcriptional terminator         sequence from T7 phage (Harrington et al., 2001).

3.1.2—Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02

Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02 is derived from pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 described above and the synthetic gene acdH from Streptomyces avermitilis optimized for Escherichia coli described below.

Synthetic Gene acdHsaO1ec

A synthetic gene of the Streptomyces avermitilis acdH gene coding for the acyl-CoA dehydrogenase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to Escherichia coli according to the supplier's matrix. The construct is cloned into supplier's pM vectors and verified by sequencing.

acdH gene sequence from Streptomyces avermitilis (AF143210) optimized for Escherichia coli: acdHsaO1ec contains the following sequence (SEQ ID No35):

atggatcatcgtctgacaccggaactggaagaactgcgtcgtaccgttga agaatttgcacatgatgagagcaccgaaaatcggcgatttctatgaacgt catgaattcccgtatgaaattgtgcgtgaaatgggtcgtatgggtctgtt tggtctgccgtttccggaagaatatggtggtatgggtggtgattatctgg cactgggtattgccctggaagaactggcacgtgagatagcagcgagcaat taccctggaagccggtgttagcctgggtgcaatgccgattcacctgtttg gcaccgatgcacagaaagcagaatggctgcctcgtctgtgtagcggtgaa attctgggtgcataggtctgaccgaaccggatggtggtagtgatgccggt gcaacccgtaccaccgcacgtctggatgaaagcaccaatgaatgggttat taatggcaccaaatgcttcattaccaatagcggcaccgatatcaccggtc tggttaccgttaccgcagttaccggtcgtaaacctgatggtaaaccgctg attagcagcattattgaccgagcggtacaccgggattaccgagcagcacc gtatagcaaagaggaggaatgcaagcgatacccgtgaactgagctagcag atgacgtgaccggcagcaaatctgctgggtgaacagggtcgtggttatgc acagtttctgcgtatcctggatgaaggtcgtattgcaattagcgcactgg caacaggtctggcacagggttgtgagatgaaagcgttaaatatgcaggcg aacgccatgcctaggtcgtaatattggtgcatatcaggcaatccagataa aatcgcagatatggaaatgaaagcccatatggcacgcgaggaggcgtgat gcagcaagccgtctggagccggtgaaccgttcaaaaaagaagcagcaatt gcaaaactgtatagcagtaccgagccgttgataatgcacgtgaagcaacc cagattcatggtggttatggattatgaatgaatatccggagcacgtatgt ggcgtgatagcaaaattctggaaattggtgaaggcaccagcgaagttcag cgtatgctgattgcacgcgaactgggtctggtgggttaa

Construction of Plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-IT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02

In this plasmid, expression of the synthetic gene is driven by a constitutive Ptrc promoter and a transcriptional terminator is added downstream of the acdHsaO1ec synthetic gene. The acdHasaO1ec synthetic gene is amplified by PCR with primers Ptrc01-acdHsaO1ec F (SEQ ID No05) and Ptrc01-acdHsaO1ec R (SEQ ID No06) using the pM vector harbouring the acdHsaO1ec synthetic gene provided by the supplier. The PCR product is digested and cloned between the BamHI and XbaI sites of the plasmid pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07 described above. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-ilvBN*(GVM20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02.

Ptrc01-acdHsaO1ec F (SEQ ID No 5) TTCTGCGGATCCgagctgttgacaattaatcatccggctcgtataatgt gtggaa GTCGACGTTAACCCTAGG taaggaggttataa atggatcatcg tctgacaccgg

with

-   -   sequence (upper case) for BamHI restriction site and extrabases,     -   sequence (bold lower case) for the trc promoter sequence (Amann         et al., 1983 and Amann et al., 1988),     -   sequence (italic upper case) for SalI, HpaI and AvrII         restriction sites,     -   sequence (underlined lower case) corresponding to RBS consensus         sequence with a PsiI restriction site,     -   sequence (italic lower case) homologous to the beginning of         acdHsaO1ec synthetic gene sequence,

Ptrc01-acdHsaO1ec R (SEQ ID No 6) ggtcgactctagaAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAG CCTTTCGTTTTATTTGATG agatct TTAACCCACCAGACCCAGTTCGCG

with

-   -   sequence (lower case) for the XbaI restriction site and         extrabases,     -   sequence (bold upper case) for T₁ transcriptional terminator         sequence from the Escherichia coli rrnB gene (Orosz et al.,         1991),     -   sequence (underlined lower case) for BglII restriction site,     -   sequence (italic upper case) homologous to the end of the         acdHsaO1ec synthetic gene sequence.

3.2—Construction of Plasmid pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07

Plasmid pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07 is derived from pBBR1MCS5 (Kovach et al., 1995) and the bkdA1-bkdA2-bkdB-lpdV operon from Pseudomonas putida ATCC-23287 coding for the branched-chain keto-acid dehydrogenase complex.

In this plasmid, expression of the bkdA1-bkdA2-bkdB-lpdV operon is driven by a constitutive Ptrc promoter and a transcriptional terminator is added downstream of the operon. The bkdA1-bkdA2-bkdB-lpdV operon is amplified by PCR with primers RBSbkdA1 F (SEQ ID No07) and lpdV-TT07 XhoI R (SEQ ID No08) using Pseudomoans putida ATCC-23287 genomic DNA. The PCR product is digested and cloned between the XbaI and XhoI sites of the pBBR1MCS5. The resulting plasmid is verified by DNA sequencing and called: pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07.

RBSbkdA1 F (SEQ ID No 7) GCCGCTCTAGAACTAGTgagctgttgacaattaatcatccggctcgtat aatgtgtggaagtcgacGTTAAC caaatacccgagcgagcg

with

-   -   sequence (upper case) for XbaI and SpeI restriction sites and         extrabases,     -   sequence (italic lower case) for the trc promoter sequence         (Amann et al., 1983 and Amann et al., 1988),     -   sequence (italic upper case) for the HpaI restriction site,     -   sequence (underlined lower case) homologous to the region         upstream of the bkdA1 gene.

lpdV-TT07 XhoI R (SEQ ID No 8) taccgggcccctcgagGCAGAAAGGCCCACCCGAAGGTGAGCCAG TCAGA TATGCAGGGCGTGGCCC

with

-   -   sequence (lower case) for ApaI and XhoI restriction sites and         extrabases,     -   sequence (bold upper case) for T7Te transcriptional terminator         sequence from T7 phage (Harrington et al., 2011),     -   sequence (underlined upper case) homologous to the end of lpdV         gene.

3.3—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02

3.3.1—Construction of Plasmid pSCB-RBS28-leuABCD-TT07-Ptrc30

Plasmid pSCB-RBS28-leuABCD-TT07-Ptrc30 is derived from pSCB (Agilent) and the leuA-leuB-leuC-leuD operon from Escherichia coli coding for 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase and two 3-isopropylmalate dehydratase respectively.

The leuA-leuB-leuC-leuD operon is amplified by PCR with primers RBS28-leuA F (SEQ ID No09) and Ptrc30-TT07-leuD R (SEQ ID No10) using E. coli MG1655 genomic DNA. The PCR product is cloned in the pSCB (Agilent). The resulting plasmid is verified by DNA sequencing and called pSCB-RBS28-leuABCD-TT07-Ptrc30.

RBS28-leuA F (SEQ ID No 09) TAACAATTTACGTAGCTCAGCCGGCACTAGTGAATTCattaaagaggaga aaGGTACC atgagccagcaagtcattattttcg

with

-   -   sequence (upper case) for SnaBI, BlpI, SpeI and EcoRI         restriction sites and extrabases,     -   sequence (bold lower case) corresponding to RBS sequence of the         pZE12-luc (Lutz et al., 1997),     -   sequence (bold upper case) for KpnI restriction site,     -   sequence (underlined lower case) homologous to the beginning of         leuA gene (83529-83505, reference sequence in the MySql         relational database (EcoGene.org))

Ptrc30-TT07-leuD R (SEQ ID No 10) tccttatacgtaTTCCACACAGTATACGAGCCGGATGATTAATCGTCAAC AGCTCgggccc GCAGAAAGGCCCACCCGAAGGTGAGCCAGgtcgac TTAA TTCATAAACGCAGGTTGTTTTGC

with

-   -   sequence (lower case) for SnaBI restriction site and extrabases,     -   sequence (bold upper case) corresponding to modified trc         promoter sequence (Amann et al., 1983 and Amann et al., 1988),     -   sequence (bold lower case) for ApaI restriction site,     -   sequence (upper case) for T7Te transcriptional terminator         sequence from T7 phage (Harrington et al., 2011),     -   sequence (underlined lower case) for Sail restriction site,     -   sequence (italic upper case) homologous to the end of leuD gene         (78848-78874, reference sequence in the MySql relational         database (EcoGene.org))

3.3.2—Construction of Plasmid pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30

Plasmid pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 is obtained from an oligonucleotide-directed mutagenesis on the pSCB-RBS28-leuABCD-TT07-Ptrc30 with primers leuA*(G462D) F (SEQ ID No11) and leuA*(G462D) R (SEQ ID No12) using pSCB-RBS28-leuABCD-TT07-Ptrc30 as template. The PCR product is digested with DpnI and transformed in competent cell. The resulting plasmid is verified by DNA sequencing and called pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30.

The mutant leuA described above confer leucine resistance in E. coli

leuA*(G462D) F (SEQ ID No 11) ggccacggtaaagatgcgcttgAtcaggtggatatcgtcgctaactac

with

-   -   base modification (upper case) to introduce the amino acid         substitution,     -   base modification (bold lower case) to introduce a BclI         restriction site.

leuA*(G462D) R (SEQ ID No 12) GTAGTTAGCGACGATATCCACCTGAtCAAGCGCATCTTTACCGTGGCC

with

-   -   base modification (lower case) to introduce the amino acid         substitution,     -   base modification (bold upper case) to introduce a BclI         restriction site.

3.3.3—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02

Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02 is derived from pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703 and the PlacIq-lacI from pTRC99A (Amersham).

In this plasmid, expression of the lacI gene is driven by its natural promoter and a transcriptional terminator is added downstream of the gene. The PlacIq-lacI is amplified by PCR with primers PlacIq F (SEQ ID No13) and lad R (SEQ ID No14) using pTRC99A (Amersham). The PCR product is digested and cloned between the BamHI and SacI sites of the pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703. The resulting plasmid is verified by DNA sequencing and called: pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02.

PlacIq F (SEQ ID No13)

tcgggcccggatcccatttacgttgacaccatcgaatgg

with

-   -   sequence (lower case) for ApaI and BamHI restriction sites and         extrabases,     -   sequence (underlined bold lower case) for the lacIq promoter         sequence

lacI R (SEQ ID No 14) ACTTAAGGAGCTC AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGA GCCTTTCGTTTTATTTGATG TACGTCACTGCCCGCTTTCCAGTCGGG

with

-   -   sequence (upper case) for Sad restriction site and extrabases,     -   sequence (underlined bold upper case) for T₁ transcriptional         terminator sequence from the Escherichia coli rrnB gene (Orosz         et al., 1991),     -   sequence (italic upper case) homologous to the end of the lad         gene.

3.3.4—Construction of Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02

Plasmid pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)-TT07-Ptrc30/RBS01-adhE2ca-TT02 is derived from pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 and pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02 described above.

In this plasmid, expression of the leucine operon is driven by an IPTG-inductible Ptrc promoter and expression of the adhE2ca gene is driven by a constitutive Ptrc promoter. A transcriptional terminator is added downstream the leucine operon. The pSCB-RBS28-leuA*(G462D)BCD-TT07-Ptrc30 is digested and cloned in the SnaBI site of the pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS01-adhE2ca-TT02. The resulting plasmid is verified by DNA sequencing and called: pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02.

3.4—Construction of the Strain 2: MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02)

Construction of a strain with increased prenol pathway flux express the ilvBN*(GMV20-22DDF)CD operon to produce 3-methyl-2-oxobutanoic acid, the leuA*(G462D)BCD operon to produce 4-methyl-2-oxopentanoate, the bkdA12B+lpdV operon from Pseudomonas putida to produce 3-methylbutanoyl-CoA, the optimized acdH gene from Streptomyces avermitilis to produce 3-methylcrotonyl-CoA and the adhE2ca gene from Clostridium acetobutylicum to produce prenol.

The pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02, pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07 and pUC19-PlacIq-lacI-TT02-Ptre01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02 plasmids are introduced by electroporation into the MG1655 strain. The presence of the three plasmids is verified and the resulting strain MG1655 (pCL1920-Ptrc01/RBS01*2-ilvBN*(GMV20-22DDF)CD-TT07-Ptrc01/RBS01*2-acdHsaO1ec-TT02) (pBBR1MCS5-Ptrc01-bkdA12B+lpdVpp-TT07) (pUC19-PlacIq-lacI-TT02-Ptrc01/OP01/RBS28-leuA*(G462D)BCD-TT07-Ptrc30/RBS01-adhE2ca-TT02) is called strain 2 (Table 1).

Example 4 Construction of Strain 4: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07)

4.1—Construction of the Strain 3: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V)

4.1.1—Construction of Strain MG1655 Ptrc30-atoB:Cm

To increase the expression level of acetyl-CoA acetyltransferase atoB, a constitutive artificial trc promoter is added upstream atoB gene into the strain MG1655 pKD46 according to Protocol 1, except that primers Ptrc01/OP01-atoB F (SEQ ID No15) and Ptrc30-atoB R (SEQ ID No16) are used to amplify the chloramphenicol resistance cassette from pKD3 plasmid.

Chloramphenicol resistant recombinants are selected. The presence of the artificial promoter Ptrc30 and the insertion of the resistance cassette are verified by PCR with primers atoB F (SEQ ID No17) and atoB R (SEQ ID No18) (Table 2) and by DNA sequencing. The resulting strain is called MG1655 Ptrc30-atoB:Cm.

Ptrc01/OP01-atoB F (SEQ ID No 15) GCATCACTGCCCTGCTCTTCTCCGGTGTCATTTTCGTCATTGGTTTAA CGCTGTTCTGACGGCACCCCTACAAACAGAAGGAATATAAACTGGCTC ACCTTCGGGTGGGCCTTTCTGC TGTAGGCTGGAGCTGCTTC

with

-   -   sequence (upper case) homologous to sequence upstream of the         atoB gene (2324042-2324130 reference sequence in the MySql         relational database (EcoGene.org))     -   sequence (underlined upper case) for T7Te transcriptional         terminator sequence from phage T7 (Harrington et al., 2001)     -   sequence (italic upper case) corresponding to the primer site 2         of plasmid pKD3 (Datsenko and Wanner, 2000)

Ptrc30-atoB R (SEQ ID No 16) CCGATAGCAGTACGTACCGCACTGACGATGACACAATTTTTCATT TAT AACCTCCTTA TTCCACACAGTATACGAGCCGGATGATTAATCGTCAAC AGCTCCATGGTCcatatgaatatcctccttag

with

-   -   sequence (bold upper case) homologous to sequence of the atoB         gene (2324131-2324174, reference sequence in the MySql         relational database (EcoGene.org))     -   sequence (italic upper case) corresponding to RBS consensus         sequence with a PsiI restriction site,     -   sequence (underlined upper case) for the trc promoter sequence         (Amann et al., 1983 and Amann et al., 1988),     -   sequence (lower case) corresponding to the primer site 1 of         plasmid pKD3 (Datsenko and Wanner, 2000).

4.1.2—Construction of Strain MG1655 ΔpdhR:Km

To delete the pdhR gene, which encodes a pyruvate dehydrogenase operon repressor, into the strain MG1655 pKD46, Protocol 1 is used except that primers DpdhR F (SEQ ID No19) and DpdhR R (SEQ ID No20) are used to amplify the kanamycin resistance cassette from pKD4 plasmid.

DpdhR R (SEQ ID No 20) gaccaattgacttcggcaagtggcttaagacaggaactcatgattc cggggatccgtcgacctgcagttcgaagttcctattctcta gaaag tataggaacttcttcaagatcccctcacgctgccgc

with

-   -   sequence (lower case) homologous to sequence of pdhR gene         (122053-122094, reference sequence in the MySql relational         database (EcoGene.org)),     -   sequence (italic lower case) corresponding to the upstream         region of pKD13-Km gene (Datsenko and Wanner, 2000),     -   sequence (bold lower case) corresponding to the FRT site and         downstream region of pKD4 plasmid (Datsenko and Wanner, 2000),

DpdhR F (SEQ ID N^(o) 19) CATCTTCTGGATAATTTTTACCAGAAAAATCACTAATTCTTTCGTTGCT CCAGTGTAGGCTGGAGCTGCTTC GAAGTTCCTATACTTTCTAGAGAATA GGAACTTCAGAGCGCTTTTGAAGCTGGGG

-   -   sequence (upper case) homologous to sequence of pdhR gene         (122839-122888, reference sequence in the MySql relational         database (EcoGene.org)),     -   sequence (italic upper case) corresponding to the downstream         region of pKD13-Km gene (Datsenko and Wanner, 2000),     -   sequence (bold upper case) corresponding to the FRT site and         upstream region of pKD4 plasmid (Datsenko and Wanner, 2000).

Kanamycin resistant recombinants are selected. The insertion of the resistance cassette is then verified by PCR with primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) (Table 2) and by DNA sequencing. The verified and selected strain is called MG1655 ΔpdhR:Km.

4.1.3—Construction of Strain MG1655 Ptrc30-atoB ΔpdhR:Km

The ΔpdhR:Km chromosomal modification is transduced into the strain MG1655 Ptrc30-atoB:Cm with a P1 phage lysate from strain MG1655 ΔpdhR:Km described above, according to Protocol 2.

Kanamycin resistant transductants are selected and the presence of ΔpdhR:Km chromosomal modification was verified by PCR with primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) (Table 2). The resulting strain is called MG1655 Ptrc30-atoB:Cm ΔpdhR:Km.

The kanamycin and chloramphenicol resistances of the above strain are removed according to Protocol 1. The loss of the kanamycin and chloramphenicol resistant cassettes is verified by PCR by using the primers pdhR F (SEQ ID No21) and pdhR R (SEQ ID No22) and atoB F (SEQ ID No17) and atoB R (SEQ ID No18) respectively (Table 2). The resulting strain is called MG1655 Ptrc30-atoB ΔpdhR.

4.1.4—Construction of Strain MG1655 lpd*(A55V):Km

To transfer the lpd*(A55V) mutation (described in WO2005073364 patent application), into the strain MG1655 Ptrc30-atoB ΔpdhR, a kanamycin resistant cassette is inserted downstream of the lpd*(A55V) gene according to Protocol 1 except that primers lpd*(A55V)_Cm F (SEQ ID No23) and lpd*(A55V)_Cm R (SEQ ID No24) are used to amplify the kanamycin resistance cassette from pKD4 plasmid.

Lpd*(A55V)_Cm F (SEQ ID N^(o) 23) cccgaaagcgaagaagaagtaatttttcgtttgccggaacatccggca attaaaaaagcggctaaccacgccgctttttttacgtctgcaagtgta ggctggagctgcttcg

with:

-   -   sequence (lower case) homologous to sequence downstream of the         lpd*(A55V) gene (129315-129405 reference sequence in the MySql         relational database (EcoGene.org)),     -   sequence (bold lower case) corresponding to the primer site 2 of         plasmid pKD4 (Datsenko and Wanner, 2000),

Lpd*(A55V)_Cm R (SEQ ID N^(o) 24) CCATACTGTCAGGCTGAATAACGAGCAACGGTCAGCAGTATGCGAACG TCTCTCTGAACGTGGAGCAAGAAGACTGGAAAGGTAAACATATGAATA TCCTCCTTAG

with:

-   -   sequence (upper case) homologous to sequence downstream of the         lpd*(A55V) gene (129491-129406 reference sequence in the MySql         relational database (EcoGene.org)),     -   sequence (bold upper case) corresponding to the primer site 1 of         plasmid pKD4 (Datsenko and Wanner, 2000).

Kanamycin resistant recombinants are selected. The insertion of the resistance cassette is then verified by PCR with primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) and by DNA sequencing. The verified and selected strain is called MG1655 lpd*(A55V):Km.

4.1.5—Construction of Strain MG1655 Ptrc30 ΔpdhR lpd*(A55V):Km

The lpd*(A55V) chromosomal modification is transduced into the strain MG1655 Ptrc30-atoB ΔpdhR with a P1 phage lysate from strain MG1655 lpd*(A55V):Km described above, according to Protocol 2.

Kanamycin resistant transductants are selected and the presence of lpd*(A55V):Km chromosomal modification was verified by PCR with primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) (Table 2). The resulting strain is called MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V):Km.

The kanamycin resistance of the above strain is removed according to Protocol 1. The loss of the kanamycin resistant cassette is verified by PCR using the primers Lpd*(A55V) ver F (SEQ ID No25) and Lpd*(A55V) ver R (SEQ ID No26) (Table 2). The resulting strain MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) is called strain 3 (Table 1).

4.2—Construction of pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 Plasmid

The pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 plasmid is derived from pUC19 plasmid (Norrander et al., 1983), adhE2 gene from Clostridium acetobutylicum coding for the bifunctional aldehyde/alcohol dehydrogenase described in patent application U.S. Ser. No. 13/169,703 and the synthetic gene mvaS from Enterococcus faecalis coding for an hydroxymethylglutaryl-CoA synthase optimized for Escherichia coli described below.

In this plasmid, expressions of both genes are driven by a constitutive trc promoter with an operator site, and a transcriptional terminator is added downstream the entire construct.

Synthetic Gene mvaSefO1ec

A synthetic gene of the Enterococcus faecalis mvaS gene coding for an 3-hydroxy-3-methylglutaryl-CoA synthase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to Escherichia coli according to the supplier's matrix. The construct is cloned into supplier's pM vectors and verified by sequencing.

mvaS gene sequence from Enterococcus faecalis (AF290092) optimized for Escherichia coli: mvaSefO1ec contains the following sequence (SEQ ID No36):

ATGACCATTGGCATCGACAAAATCAGCTTTTTTGTTCCGCCTTACTAT ATCGACATGACCGCACTGGCCGAAGCACGTAATGTTGATCCGGGTAAA TTTCATATTGGTATTGGTCAGGATCAGATGGCCGTTAATCCGATTAGC CAGGATATTGTTACCTTTGCAGCAAATGCAGCAGAAGCAATTCTGACC AAAGAAGATAAAGAAGCCATCGATATGGTTATTGTTGGCACCGAAAGC AGCATTGATGAAAGCAAAGCAGCCGCAGTTGTTCTGCATCGTCTGATG GGTATTCAGCCGTTTGCACGTAGCTTTGAAATTAAAGAAGCATGTTAC GGCGCAACCGCAGGTCTGCAGCTGGCAAAAAATCATGTTGCACTGCAT CCGGATAAAAAAGTTCTGGTTGTTGCAGCAGATATCGCCAAATATGGT CTGAATAGCGGTGGTGAACCGACCCAGGGTGCCGGTGCAGTTGCAATG CTGGTTGCAAGCGAACCGCGTATTCTGGCACTGAAAGAGGATAATGTT ATGCTGACGCAGGATATCTATGATTTTTGGCGTCCGACCGGTCATCCG TATCCGATGGTTGATGGTCCGCTGAGCAATGAAACCTATATTCAGAGC TTTGCACAGGTGTGGGATGAACATAAAAAACGTACCGGTCTGGATTTC GCAGATTATGATGCACTGGCCTTTCATATTCCGTACACCAAAATGGGT AAAAAAGCACTGCTGGCCAAAATTAGCGATCAGACCGAAGCCGAACAA GAACGTATCCTGGCACGTTATGAAGAAAGCATTATCTATAGCCGTCGT GTGGGTAATCTGTACACCGGTAGCCTGTATCTGGGTCTGATTAGCCTG CTGGAAAATGCAACCACCCTGACCGCTGGTAATCAGATTGGTCTGTTT AGCTATGGTAGCGGTGCCGTTGCAGAATTTTTCACAGGTGAACTGGTT GCAGGTTATCAGAATCATCTGCAGAAAGAAACCCATCTGGCCCTGCTG GATAATCGTACCGAACTGAGCATTGCAGAATATGAAGCAATGTTTGCA GAAACCCTGGATACCGATATTGATCAGACCCTGGAAGATGAACTGAAA TATAGCATTAGCGCCATTAATAACACCGTGCGTAGCTATCGTAACTAA

The mvaSefO1ec synthetic gene is amplified by PCR with primers RBS01*2-mvaSefO1ec-XhaI F (SEQ ID No27) and mvaSefO1ec-NheI R (SEQ ID No28) using the pM vector harbouring the mvaSefO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the XbaI and NheI sites of the pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 described in patent application U.S. Ser. No. 13/169,703. The resulting plasmid is verified by DNA sequencing and called pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02.

RBS01*2-mvaSefO1ec-XhaI F (SEQ ID N^(o) 27) gctctagaTAAGGAGGTTATAAatgaccattggcatcgac

with

-   -   sequence (lower case) for XbaI restriction site and extrabases,     -   sequence (upper case) for the RBS consensus sequence with PsiI         restriction site,     -   sequence (underlined lower case) homologous to the beginning of         mvaSefO1ec synthetic gene sequence,

mvaSefO1ec-NheI R (SEQ ID N^(o) 28) CTAGCTAGCTTAGTTACGATAGCTACGCAC

-   -   sequence (underlined upper case) for NheI restriction site and         extrabases,     -   sequence (upper case) homologous to the end of the mvaSefO1ec         synthetic gene sequence.

4.3—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07 Plasmid

The pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3457-4264-4265)-TT07 plasmid is derived from plasmid pCL1920 (Lerner and Inouye, 1990), MXAN_3757, MXAN_4264 and MXAN_4265 synthetic genes from Myxococcus Xanthus, strain DK 1622, coding for a 3-methylglutaconyl-CoA hydratase, a 3-methylglutaconyl-CoA decarboxylase subunit A and B respectively, optimized for Escherichia coli described below.

In this plasmid, these genes are organized in operon and their expressions are driven by a constitutive trc promoter with an operator site, and a transcriptional terminator is added downstream the entire construct.

4.3.1—Construction of pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec Plasmid

MXAN_3757mxO1ec Synthetic Gene

A synthetic gene of the Myxococcus xanthus MXAN_3757 gene coding for a 3-methylglutaconyl-CoA hydratase is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to Escherichia coli according to the supplier's matrix. The construct is cloned into supplier's pM vectors and verified by sequencing.

MXAN_3757 gene sequence from Myxococcus xanthus (NC_008095) optimized for Escherichia coli: MXAN_3757mxO1ec contains the following sequence (SEQ ID No37):

ATGCCTGAGTTTAAAGTTGATGCACGTGGTCCGATTGAAATTTGGAC CATTGATGGTGAAAGCCGTCGTAATGCAATTAGCCGTGCAATGCTGA AAGAACTGGGTGAACTGGTTACCCGTGTTAGCAGCAGCCGTGATGTT CGTGCAGTTGTTATTACCGGTGCCGGTGATAAAGCATTTTGTGCCGG TGCCGATCTGAAAGAACGTGCAACAATGGCCGAAGATGAAGTTCGTG CATTTCTGGATGGTCTGCGTCGTACCTTTCGTGCAATTGAAAAAAGC GATTGCGTTTTTATTGCCGCAATTAATGGTGCAGCACTGGGTGGTGG CACCGAACTGGCACTGGCATGTGATCTGCGTGTTGCAGCACCGGCAG CGGAACTGGGTCTGACCGAAGTTAAACTGGGCATTATTCCGGGTGGT GGTGGTACACAGCGTCTGGCACGTCTGGTTGGTCCGGGTCGTGCAAA AGATCTGATTCTGACCGCACGTCGTATTAATGCAGCAGAAGCATTTA GCGTTGGTCTGGCAAATCGCCTGGCACCGGAAGGTCATCTGCTGGCA GTTGCCTATGGTCTGGCCGAAAGCGTTGTTGAAAATGCACCGATTGC AGTTGCAACCGCCAAACATGCAATTGATGAAGGCACCGGTCTGGAAC TGGATGATGCACTGGCCCTGGAACTGCGTAAATATGAAGAAATTCTG AAAACCGAAGATCGCCTGGAAGGCCTGCGTGCATTTGCAGAAAAACG TGCACCGGTGTATAAAGGTCGTTAA

The MXAN_3757mxO1ec synthetic gene is amplified by PCR with primers Ptrc01/RBS01*2-MXAN_3757mxO1ec-SacI F (SEQ ID No29) and MXAN_3757 mxO1ec-KpnI R (SEQ ID No30) using the pM vector harbouring the MXAN_3757mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the Sad and KpnI sites of the pCL1920. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec.

Ptrc01/RBS01*2-MXAN_3757mxO1ec-SacI F (SEQ ID No29)

with

Ccgagctcgagctgttgacaattaatcatccggctcgtataatg tgtggaaGTCGACGTTAACACGCGTtaaggaggttataa ATGCC TGAGTTTAAAGTTG

-   -   sequence (lower case) for SacI restriction site and extrabases,     -   sequence (underlined lower case) for the trc promoter sequence         (Amman et al., 1983),     -   sequence (upper case) for SalI, HpaI and MluI restriction sites,     -   sequence (bold lower case) corresponding to RBS consensus         sequence with a PsiI restriction site,     -   sequence (italic upper case) homologous to the beginning of         MXAN_3757mxO1ec synthetic gene sequence,

MXAN_3757mxO1ec-KpnI R(SEQ ID No30)

with

CGGGGTACCTTAACGACCTTTATACACCG

-   -   sequence (underlined upper case) for KpnI restriction site and         extrabases,     -   sequence (upper case) homologous to the end of the         MXAN_3757mxO1ec synthetic gene sequence.

4.3.2—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) Plasmid

pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) plasmid is derived from pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec described above and MXAN_4264mxO1ec synthetic gene described below.

MXAN_4264mxO1ec Synthetic Gene

A synthetic gene of the Myxococcus xanthus MXAN_4264 gene coding for a 3-methylglutaconyl-CoA decarboxylase subunit A is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to Escherichia coli according to the supplier's matrix. The construct is cloned into supplier's pM vectors and verified by sequencing.

MXAN_4264 gene sequence from Myxococcus xanthus (NC_008095) optimized for Escherichia coli: MXAN_4264mxO1ec contains the following sequence (SEQ ID No38):

ATGAAAACCGCACGTTGGTGTAGCCTGGAAGAAGCAGTTGCAAGCAT TCCGGATGGTGCAAGCCTGGCAACCGGTGGTTTTATGCTGGGTCGTG CACCGATGGCACTGGTTATGGAACTGATTGCACAGGGTAAACGTGAT CTGGGTCTGATTAGCCTGCCGAATCCGCTGCCAGCAGAATTTCTGGT TGCCGGTGGTTGTCTGGCACGTCTGGAAATTGCATTTGGTGCACTGA GCCTGCAAGGTCGTGTTCGTCCGATGCCGTGTCTGAAACGTGCAATG GAACAGGGCACCCTGGCATGGCGTGAACATGATGGTTATCGTGTTGT TCAGCGTCTGCGTGCAGCAAGCATGGGTCTGCCGTTTATTCCGGCAC CGGATGCAGATGTTAGTGGCCTGGCACGTACCGAACCGCCTCCGACC GTTGAAGATCCGTTTACAGGTCTGCGTGTTGCAGTTGAACCGGCATT TTATCCGGATGTTGCCCTGCTGCATGCACGTGCCGCAGATGAACGTG GTAATCTGTATATGGAAGATCCGACCACCGATCTGCTGGTTGCGGGT GCAGCAAAACGTGTTATTGCAACCGTGGAAGAACGTGTTGCAAAACT GCCTCGTGCAACCCTGCCTGGTTTTCAGGTTGATCGTATTGTTCTGG CACCGGGTGGTGCCCTGCCGACCGGTTGTGCAGGTCTGTATCCGCAT GATGATGAAATGCTGGCACGTTATCTGAGCCTGGCAGAAACCGGTCG TGAAGCCGAGTTTCTGGAAACCCTGCTGACCCGTCGTGCAGCATAA

The MXAN_4264mxO1ec synthetic gene is amplified by PCR with primers RBS01*2-MXAN_4264mxO1ec-SmaI F (SEQ ID No31) and MXAN_4264mxO1ec-BamHI R(SEQ ID No32) using the pM vector harbouring the MXAN_4264mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the SmaI and BamHI sites of the pCL1920-Ptrc01/RBS01*2-MXAN_3757mxO1ec. The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264).

RBS01*2-MXAN_4264mxO1ec-SmaI F (SEQ ID No31)

with

cggggggtacccccgggTAAGGAGGTTATAAATGAAAACCGCACGTTGGTG

-   -   sequence (lower case) for KpnI and SmaI restriction sites and         extrabases,     -   sequence (bold upper case) for RBS consensus sequence with a         PsiI restriction site,     -   sequence (upper case) homologous to the beginning of         MXAN_4264mxO1ec synthetic gene sequence,

MXAN_4264mxO1ec-BamHI R(SEQ ID No32)

with

GCTCTAGAGGATCCTTATGCTGCACGACGGGTCAG

-   -   sequence (underlined upper case) for BamHI and XbaI restriction         sites and extrabases,     -   sequence (upper case) homologous to the end of the         MXAN_4264mxO1ec synthetic gene sequence.

4.3.3—Construction of pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265) Plasmid

pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265) plasmid is derived from pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264) described above and MXAN_4265mxO1ec synthetic gene described below and a transcriptional terminator.

Synthetic Gene MXAN_4265mxO1ec

A synthetic gene of the Myxococcus xanthus MXAN_4265 gene coding for a 3-methylglutaconyl-CoA decarboxylase subunit B is synthesized by Invitrogen. The codon usage and GC content of the gene is adapted to Escherichia coli according to the supplier's matrix. The construct is cloned into supplier's pM vectors and verified by sequencing.

MXAN_4265 gene sequence from Myxococcus xanthus (NC_008095) optimized for Escherichia coli: MXAN_4265mxO1ec contains the following sequence (SEQ ID No39):

ATGAGCGCAACCCTGGATATCACACCGGCAGAAACCGTTGTTAGCCT GCTGGCACGTCAGATTGATGATGGTGGTGTTGTTGCAACCGGTGTTG CAAGTCCGCTGGCAATTCTGGCCATTGCAGTTGCACGTGCCACCCAT GCACCGGATCTGACCTATCTGGCATGTGTTGGTAGCCTGGACCCGGA AATTCCGACCCTGCTGCCGAGCAGCGAAGATCTGGGTTATCTGGATG GTCGTAGCGCAGAAATTACCATTCCGGACCTGTTTGATCATGCACGT CGTGGTCGTGTTGATACCGTTTTTTTTGGTGCAGCCGAAGTTGATGC CGAAGGTCGTACCAATATGACCGCAAGCGGTAGTCTGGATAAACCGC GTACCAAATTTCCTGGTGTTGCCGGTGCAGCAACCCTGCGTCAGTGG GTTCGTCGTCCGGTTCTGCTGGTTCCGCGTCAGAGCCGTCGTAATCT GGTTCCGGAAGTTCAGGTTGCCACCACCCGTGATCCGCGTCGTCCTG TTACCCTGATTAGCGATCTGGGTGTTTTTGAACTGGGTGCAAGCGGT GCACGTCTGCTGGCTCGCCATCCGTGGGCAAGCGAAGAACATATTGC AGAACGTACCGGTTTTGCATTTCAGGTTAGCGAAGCACTGAGCGTTA CCAGCCTGCCGGATGCACGTACCGTTGCAGCAATTCGTGCAATTGAT CCGCATGGTTATCGTGATGCACTGGTTGGTGCATAA

The MXAN_4265mxO1ec synthetic gene is amplified by PCR with primers RBS01*2-MXAN_4265mxO1ec-XbaI F (SEQ ID No33) and MXAN_4265mxO1ec-PstI R (SEQ ID No34) using the pM vector harbouring the MXAN_4265mxO1ec synthetic gene providing by the supplier. The PCR product is digested and cloned between the XbaI and PstI sites of the pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264). The resulting plasmid is verified by DNA sequencing and called pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265).

RBS01*2-MXAN_4265mxO1ec-XbaI F (SEQ ID No33)

with

gctctagaTAAGGAGGTTATAAATGAGCGCAACCCTGGATATC

-   -   sequence (lower case) for XbaI restriction site and extrabases,     -   sequence (bold upper case) for RBS consensus sequence with a         PsiI restriction site,     -   sequence (upper case) homologous to the beginning of MXAN₁₃         4265mxO1ec synthetic gene sequence,

MXAN_4265mxO1ec-PstI R (SEQ ID No34)

with

  GCCAAGCTTCTGCAG GCAGAAAGGCCCACCCGAAGGTGAGCCAGgtat acTTATGCACCAACCAGTGCATC

-   -   sequence (underlined upper case) for HindIII and PstI         restriction sites and extrabases,     -   sequence (bold upper case) for T7Te transcriptional terminator         sequence from T7 phage (Harrington et al., 2011),     -   sequence (lower case) for BstZ17I restriction site,     -   sequence (upper case) homologous to the end of the         MXAN_4265mxO1ec synthetic gene sequence.

4.4—Construction of Strain 4: MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07)

Construction of a strain with increased prenol pathway flux expressing the atoB gene to produce acetoacetyl-CoA, the optimized mvaS gene from Enterococcus faecalis to produce 3-hydroxy-3-methylglutaryl-CoA, the optimized MXAN(3757-4264-4265) operon from Myxococcus xanthus to produce 3-methylcrotonyl-CoA and the adhE2ca gene from Clostridium acetobutylicum to produce prenol.

The pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02 and pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07 plasmids are introduced by electroporation into the strain 3 (Table 1). The presence of the two plasmids is verified and the resulting strain MG1655 Ptrc30-atoB ΔpdhR lpd*(A55V) (pUC19-Ptrc01/OP01/RBS01-adhE2ca-RBS01*2-mvaSefO1ec-TT02) (pCL1920-Ptrc01/RBS01*2-MXANmxO1ec(3757-4264-4265)-TT07) is called strain 4 (Table 1).

Example 5 Culture of the Above Described Prenol Production Strains on Glucose

Production strains 1, 2, 3 and 4 are evaluated in small Erlenmeyer flasks using modified M9 medium (Anderson, 1946, Proc. Natl. Acad. Sci. USA 32:120-128) that is supplemented with 10 g·L⁻¹ MOPS and 10 g·L⁻¹ glucose and adjusted to pH 6.8.

A 5 mL preculture is grown at 37° C. for 6.5 hours in a mixed medium (10% LB medium (Sigma 25%) with 2.5 g·L⁻¹ glucose and 90% minimal medium described above). It is used to inoculate a 50 mL culture to an OD₆₀₀ of 0.1 in minimal medium. When necessary, antibiotics are added at concentrations of 50 mg·L⁻¹ for ampicillin and spectinomycin and 10 mg·L⁻¹ for gentamycin. The temperature of the cultures is 37° C. When the culture reaches an OD₆₀₀ of 7 to 9, extracellular metabolites are analyzed using HPLC with refractometric detection (organic acids and glucose). Production of prenol is determined by GC/MS.

The strains 2 and 4 produce prenol with a concentration ranging between 0.001 and 100 mM whereas strains 1 and 3 do not produce it.

PATENT REFERENCES

-   WO 2009/006429 -   WO 2009/076676 -   WO 2010/031076 -   US 2010/0216958 -   WO 2010/031076 -   WO 2008/052973 -   WO 2008/052595 -   WO 2008/040387 -   WO 2007/144346 -   WO 2007/141316 -   WO 2007/077041 -   WO 2007/017710 -   WO 2006/082254 -   WO 2006/082252 -   WO 2005/111202 -   WO 2005/073364 -   WO 2005/047498 -   WO 2004/076659

NON-PATENT REFERENCES

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The invention claimed is:
 1. Method for fermentative production of prenol, comprising culturing a recombinant microorganism from Enterobacteriaceae family in a culture medium comprising a source of carbon, wherein in said microorganism, the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA as intermediate product, that is converted into prenol by action of an enzyme with alcohol dehydrogenase and aldehyde dehydrogenase activity.
 2. Method according to claim 1, wherein the enzyme is an alcohol-aldehyde dehydrogenase (AdhE).
 3. Method according to claim 2, wherein the AdhE enzyme is heterologous.
 4. Method according to claim 3, wherein the AdhE enzyme has a specificity for the substrate 3-methylcrotonyl-CoA.
 5. Method according to claim 2, wherein the AdhE enzyme is AdhE2 from Clostridium acetobutylicum.
 6. Method according to claim 1, wherein the biosynthesis pathway of 3-methyl-crotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 4-methyl-2-oxopentanoate and 3-methylbutanoyl-CoA.
 7. Method according to claim 6, wherein at least one of the following enzymes is overexpressed: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase.
 8. Method according to claim 7, wherein two enzymes are overexpressed: an heterologous branched chain keto acid dehydrogenase complex and an heterologous acyl-CoA dehydrogenase.
 9. Method according to claim 1, wherein the biosynthesis pathway of 3-methyl-crotonyl-CoA from pyruvate and acetyl-CoA includes the following intermediate products: 3-hydroxy-3-methylglutaryl-CoA and 3-methylglutaconyl-CoA.
 10. Method according to claim 9, wherein at least one of the following enzymes is overexpressed: an acetyl-CoA C-acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, a 3-methylglutaconyl-CoA decarboxylase.
 11. Method for the fermentative production of prenol according to claim 1, wherein the microorganism is from the species Escherichia coli or Corynebacterium glutamicum.
 12. Method for the fermentative production of prenol according to claim 1, comprising isolation of the prenol from the culture medium.
 13. Method for production of isoprene, comprising: culturing a recombinant microorganism from Enterobacteriaceae family in a culture medium comprising a source of carbon, wherein in said microorganism, the prenol biosynthesis pathway comprises 3-methylcrotonyl-CoA as intermediate product that is converted into prenol by action of an enzyme with alcohol dehydrogenase activity and aldehyde dehydrogenase activity, and performing chemical dehydration of bio-produced prenol into isoprene.
 14. A genetically modified microorganism for fermentative production of prenol according to claim 1 wherein said microorganism overexpresses at least one enzyme selected from the group consisting of: an acetolactate synthase, a keto-acid reductoisomerase, a dihydroxy-acid dehydratase, a 2-isopropylmalate synthase, a 2-isopropylmalate hydrolyase, a 3-isopropylmalate dehydrogenase, a branched chain keto acid dehydrogenase complex and an acyl-CoA dehydrogenase, an acetyl-CoA C-acetyltransferase, a HMG-CoA synthase, a 3-methylglutaconyl-CoA hydratase, and a 3-methylglutaconyl-CoA decarboxylase. 